Notch signaling is essential for the development of T cell progenitors through the interaction of NOTCH1 receptor on their surface with the ligand, Delta-like 4 (DLL4), which is expressed by the thymic epithelial cells. Notch signaling is quickly shut down once the cells pass β-selection, and CD4/CD8 double positive (DP) cells are unresponsive to Notch. Over the past two decades a number of papers reported that over-activation of Notch signaling causes T cell acute lymphoblastic leukemia (T-ALL), a cancer that prominently features circulating monoclonal CD4/CD8 double positive T cells in different mouse models. However, the possible outcomes of Notch over-activation at different stages of T cell development are unknown, and the fine timing of Notch signaling that results in T-ALL is poorly understood. Here we report, by using a murine model that ectopically expresses DLL4 on developing T cells, that the T-ALL onset is highly dependent on a sustained Notch activity throughout the DP stage, which induces additional mutations to further boost the signaling. In contrast, a shorter period of Notch activation that terminates at the DP stage causes a polyclonal, non-transmissible lymphoproliferative disorder that is also lethal. These observations resolved the discrepancy of previous papers on DLL4 driven hematological diseases in mice, and show the critical importance of the timing and duration of Notch activity.  The Notch pathway is highly conserved in multicellular eukaryotes and essential in controlling spatial patterning, morphogenesis and homeostasis in embryonic and adult tissues ,2. Hes and Hey family members, Deltex-1 or Myc, depending on cell type and context [The Notch pathway involves five ligands, four NOTCH receptors, and sequential proteolytic processing of the ligand-bound receptors to generate active Notch intracellular domain (NICD), a process in which the proteolytic activity of γ-secretase is crucial ,3. We ha context . The thymus provides a unique environment for T cell development . T cell NOTCH was discovered as the T cell receptor partner of a chromosomal translocation that resulted in T-ALL [TCR: NOTCH1 translocation; despite extensive analyses, no other mutations in the Notch pathway have been linked to T-ALL in human patients [In mammals, in T-ALL . Since tin T-ALL -17, as ain T-ALL ,19, or ein T-ALL ,21. T-ALin T-ALL ,23, within T-ALL ,26 . Howpatients ,28.Two laboratories reported the reconstitution of mice with bone marrow cells ectopically expressing DLL4 ,30. SurpIn this report, we present two new mouse models. One is called Tg8, in which DLL4 is ectopically expressed, under the transcriptional control of the TCRα, on the surface of developing and mature T cells beginning at the DP stage. All Tg8 mice succumb to T-ALL at a young age. The second mouse model is Tg8 crossed with Presenilin conditional (floxed) knock-out and CD4-cre mice (Tg8 PS KO CD4-Cre). In this model, Notch signaling is genetically abrogated at the DP stage. These DP cells do not become transformed and T-ALL does not occur. However, due to ectopic Notch signaling on precursors outside the thymus, there is an uncontrolled accumulation of polyclonal DP cells that results in massively enlarged secondary lymphoid organs. These results define an exquisite developmental window for Notch signaling effects, and help explain the discrepancy between the previous reports on DLL4 induced hematological diseases ,30.All procedures were approved by New York University’s Institutional and Animal Care Use Committee (IACUC).TCRα construct [Tg8 mice were generated as described, using the same MBP-specific onstruct . TCRα DNA preparation, by microinjection into C57BL/6 fertilized eggs.Tg8 mice and control Tg5 mice were generated with exactly the same PS1fl/fl PS2-/- (PS KO) CD4-Cre mice were previously described [Thymocytes and splenocytes were stained and run through BD LSRII. Data were analyzed with the FloJo software (Tree Star). Antibody list is provided below.  BD Pharmingen BrdU Flow Kits were used for BrdU staining. Mice were injected i.p with 1mg of BrdU in PBS and sacrificed 4 hours later. Single cell suspensions were prepared form thymus, spleen, and mesenteric LN. For PI staining, we followed established procedures . RNA was extracted using Trizol Reagent (Invitrogen) according to manufacture’s instructions. Total RNA was reverse transcribed into cDNA using SuperScript Reverse Transcriptasae II (Invitrogen). Primers are listed below.cDNA was synthesized from Tg8 pre-tumoral and tumoral spleens. PCR primer listed below.Dll4 shRNA was ligated into pQXIP-GFP. The same amount of empty vector was transfected as a control. BM cells were cultured in Optimen with SCF, FLT3L, IL6 and IL7 for 24 hrs before infection. Virus supernatants were collected and added to BM cultures. Two days later, 5x106 GFP+ cells were injected i.v into RAG1-/- recipients. anti-CD4 , anti-CD8 , anti-DLL4 monoclonal antibody (mAb) YM152F: courtesy of Dr. M. Yan, Genentech; final staining concentration: 1 μg/mL, Goat F(ab’)2 anti-human Ig(gamma)-PE as secondary antibody , anti-Notch1 , anti-NICD  . For intFoxN1 gene. After PCR, the product was digested with Bsaj 1 for 2 hours at 60°C. The sample was analyzed by electrophoresis in a 4% Nusieve agarose gel. When indicated, CD4 cells were purified by MACS using Miltenyi reagents and a Vario MACS apparatus  following the manufacturers’ instructions.PCR was performed to determine the polymorphism in the For real-time PCRCGCCAGGAAACTCTCTCATC; R: TCATTTTGCTCGTCTGTTCGDll4 F: -1 F: TGTCTGCCTTCTCTAGCTTGG; R: GCGAAGGGCAAGAATAAATGHesCACTTGAAGATGCTCCATGC; R: TTCCCGAAACCCAATACTCCHey1 (Hrt-3) F: Deltex-1 F: GAGGTCCACCAGCGTCAG; R: GCCAGTGCCATTCAAGTTCTNotch1 F: CATCCGTGGCTCCATTGTCTACC; R: AGGCTCCACCGGCTCACTCTTNotch3 F: TTCCCAGCGAGCATCCTTATTTGA R: AGTTGCTGGGCTAGGTGTTGAGTCFor Notch1 and Fbw7 sequencingNotch1 CCTCTTTGATGGCTTCGACT; R: AGCACCATCTGAGGCATTCT. Exon 25-29 F: GTGAGCTCGGCAGCCAAT; R: CCAGGAAAATCAAGGCTCTG. Exon 34 3’F: AGCACTGGCTGGAGGTAGC; R: GAGGATGGCAGTGATGTGG. Exon 34 5’ F: Fbw7GGCAACCGCATAGTTAGTGG; R: GAGATCCACTCACCACATGGExon 9-10 F:CTGGGAATGCAGATTCTACAG; R: TATTGTCTACACAGTTGGACExon 12 F: Ptcra (pre-Tα)a F: AATAGATCTCTACCATCAGGCATCGCTAATCCGCGGCTACTGGAGGTGCTGGCCCGCR: AATAGATCTCTACCATCAGGGGAATCTPtcra (pre-Tα)b F: R: AATCCGCGGCTACTGGAGGTGCTGGCCCGCTCR Cα F: TGTTCACCGACTTTGACTCC; R: TGGCGTTGGTCTCTTTGAAGEvery experiment had multiple repeats (>3 times) with at least 3 animals each time. Statistical analysis were conducted with GraphPad Prism software. The Student's unpaired t test was used as statistical test for significance. p values < 0.05 was considered significant. Error bars denote ± SEM.+CD8+ double positive or DP) to different degrees (Tg8 mice were generated from the making of a series of myelin basic protein - specific T cell receptor (MBP-TCR) transgenic mice. The MBP-TCRα genomic construct is a 40 kb Va4-Ja48 and contains promoter, enhancer and silencer elements required for tissue specific expression in the T cell lineage. The construct was inserted at random sites of the genome to generate a series of Transgenic (Tg) mouse lines. In one line, which was named Tg8, the construct was inserted on Chromosome 2. We determined that the exact insertion site had 100 percent correlation with probes D2Mit164 and D2Mit423 (MIT/Whitehead Genome Center), located in chromosome 2 at 71 cM and 68.9 cM. Interestingly, we observed spontaneous T cell lymphomas (TCL) in all Tg8 mice 1A, but  degrees S1B. The degrees 1B, indileft and central). The size of the TCRα transcript was not altered by the insertion (right). Only one copy of the TCRα transgene was inserted in Tg8 DNA  from Tg8 mice, higher than the positive control of Dll4 expression, vascular endothelial cells  cells, began at the DP stage and was maintained in both CD4 single positive (SP) and CD8 SP cells . Preceding surface expression, Dll4 mRNA was expressed at the DN4 stage . In pre-tumoral spleens, red pulp and white pulp zones were readily found, and the white pulp retained well-defined T and B cell zones (+CD8+ (DP) cells in circulating blood and spleens of Tg8 animals, as assessed by flow cytometry , and Ptcra (Pre-Tα) undergoes a profound reduction as cells transition from the DN to the DP stage. However, in Tg8 cells, expression of Notch pathway target genes remained high at the DP stage , and reconstituted irradiated RAG1-/- mice with HSC infected with either shDll4 or the empty vector. Since pre-tumoral Tg8 mice gradually accumulate DP cells in the blood, we analyzed the percentage of DP cells among the CD4+ T cells in blood every 10 days as a way to measure progression of the disease. There was a clear delay in the appearance of DP cells in mice reconstituted with shDll4-infected HSC compared to those with empty vector-infected HSC  [PS1fl allele . In contrast to Tg8 PS KO CD4-Cre mice, 19 of 19 Tg8 littermates that either lacked CD4-Cre or were PS1fl/+ developed TCL. Interestingly, Tg8 CD4-Cre PS1fl/+ PS2-/- mice showed a slight delay in tumor onset compared to Tg8 CD4-Cre PS1+/+ PS2-/- mice, probably due to lower Notch activation  with a retroviral construct harboring a short hairpin (sh) against cted HSC 4A, indilly lost S2E. TheCD4-Cre) , abrogattivation 4B. Dll4 gene on the surface of T cells leads to Notch pathway activation, causing T-ALL. Taken together, these results demonstrate that expression of a functional We took advantage of the complete penetrance of T-ALL in Tg8 mice to study the pre-tumoral events. First, we determined that the DP cells circulating in young Tg8 mice were polyclonal, while the TCL were always clonal. Thus, at some point a DP clone gains the advantage over the remaining DP cells. We therefore studied the polyclonal to clonal  transition in 13 Tg8 mice, all of which developed TCL. By Vβ staining (6 of 13 TCL) and Vβ PCR (the remaining 7 TCL), we observed that all the Tg8 tumors expressed a single Vβ 5B.Hes-1 and Deltex-1 than the polyclonal DP cells in the same mice . This result suggested that the lymphoproliferative disease in Tg8 PS KO CD4-Cre mice was not cancer, but another form of lymphoproliferation. To test this hypothesis, we transferred DP cells from Tg8 TCL (at 4 months of age) or Tg8 PS KO CD4-Cre (at 8 months of age) mice into nude recipients. After 3 to 4 weeks, all recipients of Tg8 TCL died of T-ALL , transferrable disease. The second disease is a non-clonal, non-transferrable lymphoproliferative disease with a much slower progression than T-ALL, but ultimately also lethal. T-ALL was the unavoidable outcome of Tg8 mice, a consequence of continuous Notch signaling induced by DLL4 expression on all T cells, while in WT thymus DLL4 expression is restricted to cortical epithelial cells (cTEC). Our results strongly support the notion that the same DLL4-triggered Notch pathway, which is essential for T cell generation at the DN stage, is terribly harmful to the host if not shut down at the DP stage as we forced in the Tg8 PSKO CD4-cre. Tg8 mice display elevated NOTCH1 surface expression and its activated form (NICD) only in DP cells, and, accordingly, DP cells displayed the highest expression of Notch target genes. Compared to the corresponding T cell populations in WT mice, Tg8 mice display elevated expression of surface NOTCH1 and the activated form NICD only in DP cells, and, accordingly, DP cells displayed the highest expression of Notch target genes. In addition, Tg8 DP cells were able to transfer the disease when injected in nude mice while SP cells were not. Taken together, out data suggest that DP cells are the ones at the origin of T-ALL of Tg8 mice.Notch1 or Fbw7 that have been previously associated with 50-60% of human T-ALL cases. However, 3 out 18 Tg8 lymphomas could be accounted for by mutations in PEST domain Ser residues that are associated with stabilization of NICD [T-ALL in humans and experimental animals has been correlated with increased Notch signaling, and T-ALL in Tg8 mice is no exception to this rule. There are however, new features revealed by the Tg8 T-ALL model. First, the expression of the Notch ligand DLL4 on T cells, by itself, is sufficient to drive T-ALL in all mice with a short pre-tumoral latency. In no case previously known was DLL4 expression restricted to T cells. Instead, others used Dll4-transduced bone marrow cells ,30. Seco of NICD . It remaPS1 (on a PS2 KO background). Lack of Notch signaling at the DP stage eliminated malignant transformation. In these Tg8 PS KO CD4-Cre mice, DP T cells were constantly being produced due to DLL4-triggered Notch signaling of DN cells, but, without Notch signaling on DP cells, there was no transformation to a clonal, malignant state. These observations have an implication on regular feed back mechanisms to control T cell generation. In normal situations, T cell development up to the DP stage is controlled by the limited expression of DLL4 by cTEC, but in Tg8 and Tg8 PS KO CD4-Cre mice, there is abundant DLL4 present on the surface of all T cells. Our results in nude animals, where Tg8 CD4 SP cells but not WT CD4 cells promoted the development of DP cells of nude origin clearly demonstrates that this DLL4-induced pathway does not require a thymus, and that T cells expressing DLL4 are highly competent to induce T cell development in vivo up to the DP stage. The polyclonal lymphoproliferative disease only occurred in Tg8 PS KO CD4-Cre mice in which Notch signaling on T cells was abrogated at the DP stage due to the CD4-Cre-mediated deletion of Human T-ALL has a more complex pattern with DN-like and SP-like tumors observed and Notch1 mutations at different T-cell maturation stages. In our model, DN cells are similar in WT and Tg8 mice in regard to Notch signaling, as seen by Hes1 and Hrt3 expression . HoweverDll4 [Dll4-expressing animals had a non-clonal, non-transferrable lymphoproliferative disease. As retroviral expression was differentially lost, Dll4 expression may have been shut down earlier in the case of the Dorsch et al study, and be more persistent in the T cell lineage in the Yan et al study. Our data helps explain the conflicting reports using bone marrow cells retrovirally transduced with Dll4 ,30. WhilFinally, our findings in Tg8 mice also offer a mechanism for the phenomenon of donor cell leukemia. In these cases, following allogeneic hematopoietic stem cell transplantation, the host environment induces leukemia in healthy donor cells. Depending on the study, it is estimated that between 0.1% and 5% of the post-transplant leukemia relapses in humans are caused by donor cell leukemia -46. SincFigure S1The cellular and genetic characterization of Tg8 tumors.A) Tg8 mice accumulate clonal DP cells. Tg8 tumors are clonal as indicated by dominant usage of one Vβ in each lymphoma  comparing to WT littermates . B) More than 95% of tumors were CD4+CD8+DP but a small percentage of tumors can display different CD4 and CD8 expression patterns . C) Tg8 mice have inserted a single copy of the TCRα transgene, and the TCRα is not part of any aberrant transcript. Left panel: Southern blot of DNA extracted from Tg8, WT or Tg5 animals. The probes used are indicated in D) Diagram depicting  the TCRα construct used to generate lymphoma-prone Tg8 and lymphoma-free Tg5 mice. The position of the main enhancer is indicated by a red box. The position of the 5’ and 3’ probes used in E) Summary of the transcriptional effects of Tg8 insertion at Chromosome 2 for genes located between 100 and 250 kb on either side of the integration site. The thick line between Exd1 and Cbp22 indicates the area closest to the integration site (100 kb on each side). qPCR of the indicated genes was performed, and the ratios of relative transcription level of Tg8 tissue and WT tissue were calculated as listed in the right three columns as thymus, spleen and T lymphoma (TCL). The second and third columns to the left indicate whether the raw expression values of the samples are hardly detectable above the negative control (-), slightly above the negative control (+/-), or clearly above the negative control (+). All changes in expression ratios are non-significant. F) Transcriptional effects of Tg8 insertion on the genes located nearest the integration site (100 kb on each side). It is noteworthy that Ino80 alone occupies more than half of this region, and its promoter lies 134 kb away from the TCRα enhancer in Tg8 mice. qPCR analysis was performed on cDNA obtained from sorted T cells, sorted B cells, sorted brain endothelial cells and Langerhans cells . For T and B cells, data are presented as the ratio between the transcription in Tg8 and WT cells. For endothelial cells and Langerhans cells, the ratio is taken between these cells and WT T cells. Data are represented as mean +/- SEM. G) Normal frequency of B cells in spleen of 4 w.o pre-tumoral Tg8 mice analyzed by flow cytometry.(TIF)Click here for additional data file.Figure S2Proliferation profile of Tg8 T cells and the efficiency of knock-down experiments.A) In Tg8 mice, the Notch pathway is preferentially activated in DP cells. Shown is the surface expression of the Notch target gene CD25 in the indicated populations (color-coded). B) Four week-old Tg8 and littermate controls were injected intraperitoneally with BrdU and sacrificed 4 hours later. Thymic, splenic, and mesenteric lymph node cells were surface-stained with anti-CD4 and anti-CD8 antibodies, followed by intracellular staining with anti-BrdU antibody. The panel shows the percentage of BrdU+ cells among the populations indicated in the x axis. Data are represented as mean +/- SEM. C) Propidium iodide staining of permeabilized thymic and lymph node cells from 3 week-old Tg8 and WT littermate (n=3). D) CD4 SP splenocytes sorted from 4 week-old Tg8 were cultured and transduced with either Dll4 shRNA-pQXIP/GFP vector or the same amount of empty pQXIP/GFP vector. On day 3 the cells were analyzed for DLL4 surface expression with two gates on GFP+ and GFP- populations. MFI= 12.9 in GFP- and 28.9 in GFP+E) 4 week old Tg8 BMs were transduced with either Dll4 shRNA or empty vector, and transferred into RAG1-/- recipients. CD4 SP cells from blood were analyzed for DLL4 expression at 3 and 4 months after BM transfer. Tg8 BM transduced with empty vector was also transferred into RAG1-/- mice as positive controls for lymphoma development; WT BM was transferred into RAG1-/- mice as a negative control for lymphoma development. MFI= 15.3 (empty vector), 12.6 (shRNA after 4 months), 7.3 (shRNA after 3 months), 1.7 (WT).(TIF)Click here for additional data file.Figure S3Notch1 or Fbw7 are found in Tg8 tumors.None of the known T-ALL mutations of  Lymphomas from 18 different Tg8 mice were analyzed by cDNA sequencing. Primers were designed to amplify the designated exons of Notch1 and Fbw7. PCR products were sent for sequencing, and the data were aligned with WT as well as mutated Notch1 and Fbw7. Mutations were counted and identified.(TIF)Click here for additional data file.


Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions.One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old  Translation initiation factor eIF4E binds to the cap structure  present at the 5′end of most eukaryotic mRNAs. Through its high affinity binding to the bridge protein eIF4G, this factor participates in the mRNA recruitment for translation. eIF4G interacts with the multi-subunit complex eIF3 bringing together the mRNA and the 43 S initiation complex formed by eIF3, the ternary complex (eIF2-Met-tRNAMet-GTP), the 40 S ribosomal subunit and other initiation factors A change in initiation efficiency has a strong influence on the protein content (quantitative regulation) and the relative levels of different proteins . The cap-binding protein, eIF4E, has highly conserved amino acids across all eukaryotic organisms that interact with the 5′ cap structure of mRNAs Multiple eIF4E family members have been identified in a wide range of organisms that include plants, flies, mammals, frogs, birds, nematodes, and fish Some eIF4E proteins are required only during particular developmental stages or under particular stress conditions. For example, Caenorhabditis elegans expresses four different class I eIF4E proteins; however, only IFE-3 is essential for survival In plants, three eIF4E family members have been reported: eIF4E (class I), eIF(iso)4E , and nCBP . The eIF(iso)4E protein interacts with a particular eIF(iso)4G forming the unique plant eIF(iso)4F complex In Arabidopsis, the eIF(iso)4E transcript and protein are more abundant in roots, floral organs and tissues under development In Arabidopsis thaliana, eIF4E has three genes: eIF4E1 (At4g18940), eIF4E2 (At1g29590) and eIF4E3 (At1g 29550), whereas eIF(iso)4E has one gene (At5g35620). eIF4E1 appears to be the primary form as eIF4E2 and eIF4E3 transcripts show very low expression. Depletion of eIF(iso)4E in Nicotiana tabacum and Arabidopsis thaliana, induces higher eIF4E expression, probably to compensate its function in general translation initiation. Although no obvious developmental phenotype was reported for an eIF(iso)4E knockout (KO) mutant in A. thaliana, the absence of this protein conferred resistance to infection by several potyviruses In this work we asked whether the absence of eIF(iso)4E has a discriminatory activity in mRNA translation during early Arabidopsis thaliana plant development. The previously characterized KO insertion mutant, AteIF(iso)4E-1, hereafter called (iso)4E-1, was used in microarray transcriptional and translational profiling to compare gene expression to wild type  15 day-old plants. Significantly changed genes at the translational level were genes preferentially expressed in roots and the endomembrane system. Analysis on selected genes indicated that the PHO1 transcript is present in polyribosomes of 15 day-old seedlings, but shifted towards free ribonucleoprotein fractions in the (iso)4E-1 mutant. In addition, the (iso)4E-1 mutant primary root was consistently larger at the same developmental stage compared to WT. Under phosphate limitation stress, the PHO1 protein levels were decreased in (iso)4E-1 and increased in an eIF(iso)4E overexpressing transgenic line as compared to WT. These results suggest that the presence of both isoforms, eIF4E and eIF(iso)4E, in a particular proportion is required for selective translation during normal plant growth, as well as in the phosphate limitation stress.Arabidopsis thaliana eIF(iso)4E protein, a previously reported KO mutant, (iso)4E-1, was used dspm) bearing the glufosinate ammonium (BASTA) resistance gene into the second exon of At5g35620 rendering no transcript or protein for eIF(iso)4E. To confirm the stability of the insertion, mutant seedlings were grown in the presence of BASTA and analyzed at RNA and protein levels (eIF(iso)4E RNA or protein was detected for (iso)4E-1, whereas the eIF4E protein was increased in agreement with To access the functional relevance of the n levels . No eIF((iso)4E-2, -3, and -4, expressing eIF(iso)4E fused to a GFP tag at the carboxy terminus in the Col-0 WT background (7GTP-Sepharose ((iso)4E-2 line was selected, whereas for others the three transgenic lines were included. Additionally, a transgenic line expressing under a 35 S promoter eIF4E (4E-ϕ) fused to a GFP tag at the carboxy terminus in the Col-0 WT background was used as control for some experiments 4E-1 did not report any obvious phenotype in leaf development and morphology, plant size, flowering and fertility, inferring that the eIF4E protein is able to function in place of eIF(iso)4E. However, even though the two proteins belong to Class I and might perform partially redundant function, changes at the level of selective translation of mRNAs due to the lack of either cap-binding protein may not be ruled out (iso)4E-1 mutant seedlings was analyzed using Arabidopsis Oligonucleotide Microarray slides obtained from the University of Arizona . The developmental stage was chosen on the basis of enriched eIF(iso)4E expression in actively growing tissues Arabidopsis thaliana tissues were not available. The sedimentation profiles on 20–60% sucrose gradients 260 indicated no global differences between wild type and (iso)4E-1 mutant plants (Previous reports describing t plants . The frahttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29386), significantly changed genes in the mutant NP and P fractions were found on a cutoff of 1.5 fold either up or down. According to this criterion 1474 transcripts decreased and 1621 increased in NP, whereas 1558 transcripts decreased and 1491 increased in P fractions of (iso)4E-1 plants. However, from these only 79 transcripts decreased their presence in P with a concomitant increase in NP , the ABC transporter-like Arabidopsis thaliana multidrug resistance associated protein 11 (MRP11) with ATPase activity, the sucrose transporter 3 (SUC3) and five electron transporters . Additionally, other plasma membrane and endomembrane system proteins involved in signal transduction and post-translational modification of amino acids were abundant in this group. Genes belonging to Group II included many transcription factors and DNA-binding proteins, as well as two histone acetyl transferases (At5g56740 and At1g77540), translation initiation factor 3 k (eIF3k) and the ribosomal protein L12 (rpL12). Hence, several of the constituents of Group II may be associated with important changes at transcription level in the (iso)4E-1 KO mutant.The 79 translationally inhibited transcripts represent potential candidates to be preferentially recruited by eIF(iso)4E for their translation. Of these mRNAs, ≈33% were preferentially expressed in roots and ≈25% in inflorescences , similar(iso)4E-1 mutant, whereas 296 were repressed with a cutoff of z-score greater than 2 either up or down. Most of the mRNAs that changed corresponded to metabolism and gene information related proteins  RNA level. However, the transcriptome analysis accessed by total RNA analysis of the same plants by microarrays, indicated that 496 genes were up-regulated in the proteins . Such fiSeveral randomly selected gene IDs from Groups I and II were chosen to confirm the microarray data by real time reverse transcription PCR (qRT-PCR) on a different biological sample. As observed in Previous work with plant eIF4E and eIF(iso)4E as part of their corresponding cap-binding complexes has indicated that they might display selection on mRNA recruitment depending on the 5′UTR length and presence of secondary structure in vitro experiments performed years ago with wheat eIF4F and eIF(iso)4F complexes The 5′ and 3′ UTR regions were downloaded from The Arabidopsis Information Resource (TAIR10: 11/17/10 release) and the longest expressed sequence tag was selected for each UTR. To avoid partial 5′end sequences, further analysis subtracted the gene accessions listed in the Salk-Stanford-PGEC Arabidopsis ORF gene list (iso)4E-2 transgenic line there was an alteration on the polyribosomal distribution of some of the mRNAs shown in eIF4A was used as control mRNA apparently not affected by eIF(iso)4E levels. To evaluate more accurately the distribution of selected mRNAs along the polyribosomal profiles, the NP fraction used for microarray analysis was further divided into monosomes (M) and free ribonucleoproteins, RNPs (NP), whereas the P fraction was divided into low polyribosomes (LP) and high polyribosomes (HP). The mRNA corresponding to the phosphate transporter PHO1 was shifted to NP and M in the (iso)4E-1 null mutant whereas it increased in LP and was similar in the HP with respect to WT in the (iso)4E-2 overexpressing line. This places the PHO1 mRNA as a strong candidate for eIF(iso)4E dependent translation.To test whether in the ERF/AP2, which appeared shifted particularly toward the HP fraction in (iso)4E-1 ((iso)4E-2 overexpressing line this mRNA was increased in the M fraction as compared to the WT profile. These results suggest that the proportion of eIF(iso)4E to eIF4E proteins is relevant for in vivo selective mRNA translation.In the absence of eIF(iso)4E, the mRNAs from Group II were modestly enhanced on polysomes . Such beiso)4E-1 . On the (iso)4E-1 KO mutant, increased eIF4E protein levels have been reported PHO1 and/or ERF/AP2 factor in the polyribosomal profile of this mutant. We used a transgenic line overexpressing eIF4E to evaluate the polyribosomal distribution of these mRNAs 4E in the (iso4E)-1 null mutant is responsible for the observed downregulation of PHO1 mRNA translation.In the se mRNAs . The 4E-Arabidopsis thalianaPHO1 expression in 15 day-old WT and mutant seedlings transferred for 10 additional days to no (−Pi) or normal (+Pi) phosphate concentration in the medium. As expected, the PHO1 transcript level was increased in WT phosphate starved plants ((iso)4E-1 and (iso)4E-2, mutant lines the PHO1 mRNA levels were similar to WT in the presence of phosphate, but showed only a modest increase under Pi starvation conditions compared to WT suggesting that there might be an indirect effect of eIF(iso)4E levels on transcription or stability of PHO1 mRNA in response to phosphate starvation. We further analyzed the PHO1 protein levels by western blot in WT and eIF(iso)4E mutant plants under both, +Pi and –Pi conditions ((iso)4E-2 overexpressing mutant, but not in the (iso)4E-1 KO mutant. Using the same amount of protein from leaves, PHO1 was not detected in accordance with a previous report using the same antibody (iso)4E-1 roots the PHO1 protein was undetectable. Under normal growth conditions (+Pi) the PHO1 protein levels remained beyond the western blot sensitivity, even for the (iso)4E-2 overexpressing mutant. Taking into account these results and the previous data on PHO1 polysomal distribution by qRT-PCR (PHO1 mRNA translation.Since PHO1 is a major phosphate transporter required under phosphate (Pi) deficiency in d plants , indicatnditions . For the qRT-PCR  and S7 wArabidopsis seedling root and lower hypocotyl, its altered expression levels in AteIF(iso)4E mutants may affect the root architecture under normal growth conditions. Analysis of WT and mutant plants, indicated that 25 day-old (iso)4E-1 seedlings [grown for 15 days on Gamborg's B5 and transferred for 10 additional days on Hoagland (+Pi)] had increased primary root length by 1 cm when compared to WT ((iso)4E-2 had a shorter length by approximately 2 cm at the same time. The observed differences were statistically significant at p<0.001. The shorter root phenotype was reproducibly observed for three independent transgenic lines expressing AteIF(iso)4E under a constitutive S35 promoter, ruling out that the altered root growth might be a result of a particular transgene insertion site (Since the PHO1 protein is involved in phosphate uptake and is predominantly expressed in ed to WT . On the ion site .(iso)4E-1 or (iso)4E-2 seedlings in phosphate starvation ((iso)4E-1 than WT (p<0.001), whereas there was only a slight reduction in growth of an already shorter (iso)4E-2 root (p<0.05). Hence, the effect on root growth observed under altered eIF(iso)4E levels was surpassed by the condition of Pi deficiency.Inhibition of primary root growth is a characteristic plant phosphate starvation response arvation . StatistThe presence of multiple eIF4E family members is widespread in eukaryotes including plants. Two different proteins, eIF4E and eIF(iso)4E, have been shown to participate in plant translation initiation through cap-binding of the mRNAs and recruiting other initiation factors The quantitative RT-PCR analysis performed for a limited number of genes confirmed a wide range of the changes between NP and P fractions . These rAccording to the developmental stage at which the microarray analysis was performed, it was interesting to notice that in (iso)4E-1 most of the translationally affected transcripts represented root expression and endomembrane system proteins, whereas those translationally enhanced were inflorescence-expressed and transcription factors. Available data on eIF4E and eIF(iso)4E expression patterns are indicative of preferential, albeit not exclusive, localization of the eIF(iso)4E protein in young roots, whereas eIF4E is more ubiquitously distributed Since several root-expressed mRNAs changed their translation profile in the absence of eIF(iso)4E, a logical assumption was to look more closely to the root morphology of 15 day-old Arabidopsis WT and mutant seedlings. The previous (iso)4E-1 mutant characterization had not observed the phenotype of roots The change in specific mRNA polyribosomal distribution in (iso)4E-1, such as that corresponding to PHO1, lead us to propose that eIF(iso)4E might be particularly required during stress conditions such as phosphate deficiency, where PHO1 protein levels are crucial The availability of soil nutrients can alter root development PHO1 altered translational distribution in (iso)4E-1 and (iso)4E-2 mutants and the observed root phenotype, it may exist or not. A couple of recent publications have shown very interesting new concepts about the PHO1 role in the Pi deficiency related-phenotype and an influence of its transcript expression levels on Pi efflux and cell growth PHO1 levels, and (2) an increase in the PHO1 transcript levels in shoot induces major Pi efflux from the cell toward the vascular system with a concomitant cell growth reduction. Taking this into account, changes in PHO1 translational levels under normal growth conditions in the eIF(iso)4E mutants used in this study may have contributed, in addition to other gene expression alterations, to the root growth phenotype.Regarding a correlation between the Overall the results obtained here indicate that selective translation by altering the eIF(iso)4E levels and correspondingly its proportion relative to eIF4E is much more complex, than simply assuming that each factor specifically recruits different group of mRNAs to the ribosome. In addition to the preference of each factor for a particular 5′UTR, eIF4G or eIF(iso)4G within the eIF4F and eIF(iso)4F cap-binding complexes, other translation factors or regulatory proteins, probably account for the plasticity in the translation profile of a given mRNA in response to stress conditions.Arabidopsis thaliana ecotype Columbia (Col-0) were obtained from the Arabidopsis Biological Resources Center (ABRC) at Ohio State University. The transposon insertion mutant for the eIF(iso)4E gene, (iso)4E-1, was kindly donated by Dr.Christophe Robaglia from CNRS-CEA Université de la Mediterranée, France. Transgenic lines expressing eIF(iso)4E as GFP fusion protein under the constitutive Cauliflower Mosaic Virus (CaMV) 35 S were generated using the Gateway® cloning technology . The DNA coding region of eIF(iso)4E or eIF4E was first amplified by PCR with the appropriate primers and then cloned into pTOPO-D vector (Invitrogen). Finally, the coding region was introduced by recombination with LR clonase (Invitrogen) into the pEarleyGate103 binary vector Agrobacterium tumefaciens strain, and used to transform Col-0 plants by floral dip transformation (iso)4E-2 was selected as representative for the experiments based on mRNA expression levels (4E-ϕ) was selected for some experiments as described in results.Seeds of wild type (WT) n levels . The samAll reagents were from Sigma-Aldrich Quimica, Mexico unless otherwise stated. Arabidopsis thaliana seeds were surface-sterilized in Tween 20 (0.1%) and sodium hypochlorite 20% for 10 minutes and rinsed 5 times with sterile deionized water. The seeds were spread on sterile 0.7% (w/v) agar  containing Gamborg's B5 minimal medium with 1% sucrose. The seeds were stratified at 4°C in the dark  for two days and then grown for 15 days at 20°C under 100 µmol m-2 sec-1 light intensity in a 8/16 hr light/dark photoperiod.3, 100 µM Ca(NO3)2, 1 mM MgSO4, 25 µM Fe-EDTA, 14 µM MnCl2, 0.5 µM CuSO4, 1 µM ZnSO4, 70 lM H3BO3, 0.2 µM NaMoO4 and 10 µM NaCl  supplemented with either 1 mM (+Pi) or 10 µM (−Pi) KH2PO4.Plants used for protein analysis were grown for one week in a medium containing half-strength Murashige-Schoog (MS), 1% sucrose and 0.6% gellan. Then, seedlings were transferred to a liquid medium containing 1% sucrose, 2.5 mM KNO3, 4 mM Ca(NO3)2·H2O, 2 mM MgSO4·7H2O, 0.009 mM MnCl2·4H2O, 0.046 mM H3BO3, 0.0008 mM ZnSO4·7H2O, 0.0003 mM CuSO4·5H2O, 0.0001 mM H2MoO4·H2O, 0.005 g/ml Fe-EDTA and 0.5 mM NH4H2PO4 as a phosphate source (+Pi), or Hoagland medium including all of the above except for the 0.5 mM NH4H2PO4 which was substituted by 0.5 mM (NH4)2SO4 (−Pi) for 10 days.For root phenotype studies, seeds were grown in Gamborg's B5 solid medium for two weeks and afterwards transferred to Hoagland medium containing 6 mM KNOTo scan root development, Arabidopsis thaliana seeds were grown in square Petri dishes oriented vertically. At 20 days after germination the plants were carefully removed from the medium and the primary root length was measured using digital ruler (ImageJ). Five plants per line 4E-1 and (iso)4E-2 mutants) were tested per experiment, and the experiment was repeated 9 times. To analyze the effect of phosphate deficiency on root development, Arabidopsis seeds were germinated on Gamborg's B5 for 2 weeks and then transferred to Hoagland medium containing phosphate (+Pi) or without phosphate (−Pi) as described above. Five plants per line were tested per experiment, and the experiment was repeated 5 times. All data were analyzed by one way ANOVA with Tukey's Multiple Comparison Test using a p value<0.05.g for 15 min and clear supernatants were collected. The protein concentration was determined using Bradford reagent  and bovine serum albumin (BSA) as the standard. For cap-binding protein purification, the protocol from Total protein extracts were prepared by homogenizing frozen leaf or root material in extraction buffer  in the ratio of 2 g of tissue to 3 ml buffer. The samples were centrifuged at 13 000 Thirty micrograms of total protein were separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane , which was blocked with 5% (w/v) milk in PBS-Tween and incubated with the primary antibody overnight at 4°C. Rabbit antibodies to Arabidopsis eIF4E and eIFiso4E were kindly provided by Dr. Karen Browning  and used at 1∶10,000 dilution. Rabbit antiserum against Arabidopsis PHO1 was kindly donated by Prof. Yves Poirier  and used at 1∶1,000 dilution. The secondary antiserum was used at 1∶10,000 dilution and incubation for 1.5 h at room temperatutre. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore Corp.).Two grams of 15 day-old Arabidopsis thaliana seedlings were crushed with liquid nitrogen and homogenized in 2 ml of lysis buffer consisting of 200 mM Tris-HCl, 50 mM KAc, 25 mM MgAc, 2 mM EGTA 2, 2% PTE (10 Tridecyl Polyoxyethylene ether), 1% Nonidet P-40, now IGEPAL and 50 mg/ml cycloheximide. After the lysis, samples were centrifuged for 15 min at 12,000 g at 4°C. The supernatant was placed on 60% sucrose in gradient buffer containing 50 mM Tris-HCl, 20 mM KAc, 2 mM EGTA and 50 mg/ml cycloheximide and centrifuged in a Beckman 75Ti rotor at 50,000 rpm for 3 hours at 4°C. The pellet was dissolved in 400 µl of DEPC water, layered onto 20 to 60% sucrose continuous gradient and centrifuged in a Beckman SW40 rotor at 36,000 rpm for 2.5 h at 4°C. The gradient was fractionated with continuous monitoring of the absorbance at 260 nm.2 and homogenized in Trizol ® Reagent (Invitrogen). Chloroform was added at 1/6 (v/v), the mixture was vortexed and then centrifuged at 12,000 g for 15 min at 4°C. The colorless upper aqueous phase was carefully collected and precipitated with 1 volume of isopropanol at room temperature. After centrifugation at 12,000 g for 20 min at 4°C, the RNA pellet was washed with 75% ethanol, the ethanol removed by an additional round of centrifugation and the RNA dissolved in 50 µl of diethyl pyrocarbonate (DEPC)-treated water.For total RNA isolation, 100 mg of 15 day-old seedlings were crushed with liquid NFor RNA isolation from sucrose gradient fractions, each fraction was treated with 0.5% sodium dodecyl sulfate (SDS) and 20 ug/ml proteinase K (Sigma), then extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol [25∶24∶1 (v/v/v)] by vortexing. The mixture was centrifuged at 10,000 g for 5 min at 4°C. The aqueous phase was brought to 1.5 M with 10 M LiCl and an equal volume of cold isopropanol was added. The RNA was precipitated at −20°C for 1 h and centrifuged at 12,000 g for 15 min at 4°C. The pellet was washed with 70% ethanol and dissolved in 30 µl of DEPC-treated water.260 profile of the sucrose gradient  and polyribosomal (P) according to its electrophoretic pattern and the Agradient .http://www.arizona.edu/microarray/). For pre-hybridization, the slides were re-hydrated with water vapor at 60°C, and fixed with two cycles of UV light (1200J). After boiling for two minutes at 92°C, slides were washed with 95% ethanol for one minute and pre-hybridzed in 5× SSC, 0.1% SDS and 1% BSA for one hour at 42°C. The slides were washed and dried for further hybridization. Ten µg of RNA were used for cDNA synthesis incorporating dUTP-Alexa555 or dUTP-Alexa647 by the First-Strand cDNA labeling kit (Invitrogen). Incorporation of the fluorophores (dUTP-CY5 and dUTP-CY3) was analyzed by the absorbance at 555 nm for Alexa555 and 650 nm for Alexa647. Equal quantities of labeled cDNA were hybridized using UniHyb  to the arrays for 14 h at 42°C. Two replicate experiments were performed with RNA obtained from independent plant groups.Microarray slides were fabricated by the University of Arizona using the Qiagen-Operon Arabidospis Genome Array Ready Oligo Set (AROS V.3.0) containing 29,000 oligonucleotides . GenArise carry out a number of transformations: background correction, Lowess normalization, intensity filter, replicates analysis and selecting differentially expressed genes. The goal of genArise is to identify which of the genes show good evidence of being differentially expressed. The software identifies differentially expressed genes by calculating an intensity-dependent z-score. It uses a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point, and define a z-score where z measures the number of standard deviations from the mean: zi = [Ri – mean(R)]/sd(R), where zi is the z-score for each element, Ri is the log-ratio for each element, and sd(R) is the standard deviation of the log-ratio. With this criterion, the elements with a z-score >1.5 standard deviations would be the significantly differentially expressed genes. All detailed protocols and data can be accessed online at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29386.Acquisition and quantification of array images was performed in ScanArray 4000 with its accompanying software ScanArray 4000 from Packard BioChips. All images were captured using 65% PMT gain, 70 to 75% laser power and 10 µm resolution at 50% scan rate. For each spot the Alexa−ΔCt method described previously Two micrograms of RNA were treated with RQ1 DNase  and subjected to reverse transcription using M-MLV-RT (Invitrogen). The PCR was performed with gene-specific primers designed in the MacVector program 4E-1 mutant using The Arabidopsis Information Resource (TAIR10: 11/17/10 release). The sequences present in the Salk-Stanford-PGEC Arabidopsis ORF gene list http://mfold.rit.albany.edu), and the presence of uORFs, uAUGs, IRES-like sequences and CPE elements by UTRScan (http://www.ba.itb.cnr.it/BIG/UTRScan) and RegRNA (http://regrna.mbc.nctu.edu.tw).The 5′ and 3′ UTR regions, based on the longest expressed sequence tags, were downloaded for mRNAs translationally affected in the Figure S1Molecular characterization of eIF(iso)4E overexpressing Arabidopsis thaliana transgenic plants.(PDF)Click here for additional data file.Figure S2Protein structural modeling of eIF(iso)4E-GFP.(PDF)Click here for additional data file.Figure S3GFP localization in transgenic plants.(PDF)Click here for additional data file.Figure S4Sucrose gradient sedimentation profiles.(PDF)Click here for additional data file.Figure S5eIF(iso)4E expression patterns in different Arabidopsis thaliana tissues and developmental stages.(PDF)Click here for additional data file.Figure S6Classification of transcripts significantly changed at steady-state level in the (iso)4E-1 mutant according to the biological process where they are involved.(PDF)Click here for additional data file.Figure S7Polyribosomal distribution of selected mRNAs in an eIF4E overexpressing transgenic line.(PDF)Click here for additional data file.Figure S8Increase in PHO1 transcript level in response to phosphate deficiency.(PDF)Click here for additional data file.Figure S9Primary root length in three independent transgenic Arabidopsis lines overexpressing eIF(iso)4E.(PDF)Click here for additional data file.Table S1List of oligonucleotides used as primers for real time RT-PCR (qRT-PCR).(PDF)Click here for additional data file.Table S2List of mRNAs identified by microarray analysis that significantly decreased their levels in polyribosomes (P) and increased in non-polyribosomes (NP) in the (iso)4E-1 mutant.(PDF)Click here for additional data file.Table S3List of mRNAs identified by microarray analysis that significantly increased their levels in polyribosomes (P) and decreased in non-polyribosomes (NP) significantly in the (iso)4E-1 mutant.(PDF)Click here for additional data file.


Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the MmmHstx2 allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species  trans-acting male-specific hybrid sterility genes. We located one of the two major hybrid sterility genes to a 4.7 Mb interval on Chromosome X, showed that it controls male sterility by modulating the extent of meiotic asynapsis and using the inter-subspecific chromosome substitution strains we refuted the simple interpretation of dominance theory of Haldane's rule. A new working hypothesis posits male sterility of mouse inter-subsubspecific F1 hybrids as a consequence of meiotic chromosome asynapsis caused by the cis-acting mismatch between orthologous chromosomes modulated by the trans-acting hybrid male sterility genes.Genomes of newly emerging species restrict their gene exchange with related taxa in order to secure integrity. Hybrid sterility is one of the reproductive isolation mechanisms restricting gene flow between closely related, sexually reproducing organisms. We showed that hybrid sterility between two closely related mouse subspecies is executed by a failure of meiotic synapsis of orthologous chromosomes in F1 hybrid males. The asynapsis of orthologous chromosomes occurred in meiosis of male and female hybrids, though only males were sterile due to  It is defined as a condition where two parental forms fertile M. m. musculus and M. m. domesticus subspecies  as model organisms to study mammalian HS . When mated with Mmm wild mice trapped in Central Bohemia near Prague, these crosses produced sterile or fertile male hybrids, depending on their Hst1 alleles Hst1 was identified by the forward genetics approach as PR domain containing 9 (Prdm9) We identified the first hybrid sterility gene in mice, hybrid sterility 1 – Mmm×Mmd)×Mmd backcross predicted a minimum of four independently segregating HS loci. However, QTL analysis of the data revealed only two strong HS loci, Hst1/Prdm9 and a locus on Chr X Mmd) genetic background In a study of genetic architecture of F1 hybrid male sterility, the results of  inbred strain Mmd represented by the C57BL/6J inbred strain (hereafter B6) is controlled by the central region of Chr X . To localize the locus responsible for HS asymmetry we crossed consomic F1 females (B6.PWD-Chr X×B6) with PWD males. All 124 male offspring carried B6/PWD heterosubspecific autosomal pairs while Chr X loci were either PWD or B6, depending on the recombination breakpoints. Testes weight  and sperm count  were used as surrogate for QTL analysis of male fertility phenotypes. Their segregation localized a hybrid sterility locus to the 34.6–35.7 cM  interval with the maximum LOD score 30 and 23 at 34.8 cM for TW and SC. As shown in DXMit87 locus had small testes bellow 100 mg and little (below 106) sperm in ductus epididymis. This hybrid sterility locus causes complete sterility on F1 hybrid intersubspecific background, and we designate it hybrid sterility chromosome X 2, Hstx2. Earlier, we reported localization of the locus at lower resolution in the (PWD×B6)×B6 backcross (Dzur-Gejdosova 2012).Dobzhansky-Muller incompatibilities (DMIs) between the nascent species often result in asymmetry of HS or inviability of reciprocal F1 hybrids Hstx2, a new partial consomic strain B6.PWD-Chr X.1s was created with extended proximal interval of the Chr XPWD sequence compared to B6.PWD-Chr X.1  males, showing meiotic arrest at epithelial stage IV and to lesser degree at late pachytene/diplotene stage .The borders of the PWD sequence of the introgressed Chr Xexamined . The , RIKEN cDNA 4930447F04 gene (4930447F04Rik), SLIT and NTRK-like family, member 2 (Slitrk2), RIKEN cDNA 4933436I01 gene (4933436I01Rik), fragile X mental retardation syndrome 1 homolog (Fmr1), fragile X mental retardation 1 neighbor (Fmr1nb) and AF4/FMR2 family member 2 (Aff2) show high expression in adult testis. Of them, Aff2 is expressed pre-meiotically in spermatogonia, Fmr1 and Fmr1nb show expression in early prophase I, and Ctag2, 4930447F04Rik and Slitrk2 are expressed in meiotic and postmeiotic cells. Finally, 4933436I01Rik is expressed in haploid cells. Sorted populations of testicular cells from PWD and B6 strains and immature 14.5 dpp testes of their reciprocal hybrids did not show significant differences in relative mRNA expression levels of six meiotic and/or postmeiotic genes  confirmed the same SNPs for PWK, a closely related Mmm inbred strain. Search of miRNA sequences revealed one SNP in the seed sequence of Mir743a, changing AAAGACA in B6 to AAAGACG in PWD . Fmr1nb, another possible candidate, shows high expression in pre-pachytene spermatocytes Reproductive isolation genes in volution . In partB6Hstx2 to rescue the meiotic arrest of Mmm×Mmd F1 hybrids is subject to intrasubspecific Mmm polymorphisms. While asymmetric male sterility of (PWD×B6)F1 hybrids depends on the presence of the PWDHstx2 allele, another inbred strain derived from Mmm, known as STUS produces fully sterile F1 hybrid males with B6 mice regardless of the direction of the cross Mmm Chr XB6, we genotyped 84 test-cross males from crosses of B6 females with (PWD×STUS)F1 or (STUS×PWD)F1 males. QTL analysis of the sperm count  revealed QTLs on Chr 3, Chr 9 and Chr 13, while QTL for the testes weight mapped on Chr 3 and Chr 13  hybrid males ensured full sterility, meiotic arrest at mid-late pachynemas, almost absent diplotene spermatocytes and a lack of sperm. Immunostaining of SYCP3 and SYCP1 components of lateral and central elements of synaptonemal complexes or HORMAD2 protein revealed unsynapsed autosomes in >90% of pachynemas of both, (PWD×B6) and (B6.PWD-Chr X1s×PWD) F1 hybrid males. The super-resolution structured illumination microscopy documented irregular spots of SYCP1 on some univalents and nonhomologous synapsis and/or translocations F1 hybrid males is associated with failure of proper synapsis of homologous heterosubspecific autosomes, delay of DNA double-strand break (DSB) repair on unsynapsed autosomes and dysregulation of meiotic sex chromosome inactivation (MSCI) at the first meiotic prophase. However, full fertility and complete autosomal synapsis is restored when Chr 17 is PWD/PWD consubspecific on otherwise PWD/B6 F1 background x2 locus –E. The Hocations , resemblutosomes –E. It caMmd . Both F1 hybrids were sterile, showing no sperm (STUS×B6) or few sperm  in ductus epididymis. In both cases >90% of pachytene spermatocytes revealed multiple pairs of unsynapsed autosomes  strain combination, we checked chromosome synapsis in pachytene spermatocytes of (STUS×B6)F1 and (PWD×SCHEST)F1 hybrids. SCHEST is a wild-derived strain of utosomes , B. ThusPrdm9 and Hstx2 genes in sterile hybrid males  when compared with the other intersubspecific F1 hybrid genotypes  homologous autosomes evade asynapsis in otherwise heterosubspecific (PWD/B6) genomic background of hybrid males. The finding indicated a PWD, Chr X.1PWD or Chr X.1sPWD, respectively. We compared the efficacy of meiotic synapsis of consubspecific (PWD/PWD) Chr 17 and Chr X homologs with matching heterosubspecific pairs in (PWD×B6)F1 pachytene oocytes and oocytes from the parental controls. Using the whole-chromosome DNA FISH we also visualized Chrs 2, 16, 18, 19 and X. In (PWD×B6)F1 pachynemas Chr 2 showed the lowest incidence of asynapsis. The frequency of univalents of small autosomes 16, 17, 18 and 19 varied between 18% and 49% in asynaptic pachytene oocytes. Strikingly, Chr X displayed the highest frequency (64%) of asynapsis F1 oocytes, although the total frequency of pachynemas with asynapsis was the same as in (PWD×B6) hybrids. In (PWD×B6.PWD-Chr X.1s)F1 oocytes, 69.9 Mb of the centromeric part of Chr X was consubspecific for the PWD sequence, while the end of the chromosome, 101.4 Mb in length, was PWD/B6 heterosubspecific. Nevertheless, the partial PWD homozygosity was sufficient to reduce Chr X asynapsis from 64% down to 5.6% of pachytene oocytes F1 hybrid females were fully fertile, as were the parental controls  but not in homozygous (XX) sex controls . HoweverDrosophila hybrids  and showed that it also carries the Hstx1 locus. In contrast to Hstx1 phenotype, the intrameiotic arrest is controlled from a single Hstx2 locus on Chr X. The Hstx2/Hstx1 critical interval is overlapped by and may be identical with the 8.4 Mb QTL responsible for the sterilizing effect of Mmm Chr XPWK introgressed into the genetic background of Mmd LEWES inbred strain Ctag2, 4933436I01Rik and Fmr1nb) also occur in the present list of Hstx1/2 candidates. Moreover, the Sha2 locus of the Japanese house mouse Mus m. molossinusHstx1, maps to the same interval. Sha2 is responsible for spermiogenic arrest in B6 males carrying Chr XM.m.molossinus introgression Fmr1nb and 4933436I01Rik are expressed in the appropriate cell type during germ cell differentiation and display two and seven non-synonymous substitutions, respectively. The critical region also contains the Mir465 cluster of miRNA genes, which show a significant difference in expression between reciprocal F1 hybrids. Moreover, Mir743a carries a single SNP in its seed sequence. Admittedly, the Hstx1/Hstx2 candidate region is still too large to finalize the list of Hstx2/Hstx1 candidate genes. Recently, many ampliconic genes on the mouse and human Chr X were shown to be unique for a given species and expressed predominantly in testicular germ cells Hstx1/Hstx2 critical region is flanked by amplicons 4930527E24Rik and Xlr  F1 hybrid males displayed premeiotic block with Sertoli cells and dividing spermatogonia. The QTL analysis of a backcross population revealed two major loci, again on Chr 17 and Chr X, but with positions distal to Prdm9 and to Hstx1/Hstx2 loci, respectively The proximal part of Chr X carries loci for major incompatibilities also outside the Mmm/M. m. molossinus and MmdLaboratory crosses of wild-derived inbred strains continue to reveal new details about the genetic control and mechanistic basis of hybrid sterility, but their direct role in speciation is less clear. In an alternative approach, evolutionary biologists collect wild mice from the house mouse hybrid zone to analyze the introgression of (sub)species-specific DNA markers. Because this approach can reflect all kinds of incompatibilities restricting the gene flow, the results are usually complex, uncovering multiple regions on autosomes and Chr X. Nonetheless, at least two common themes have emerged from the field data and laboratory crosses, namely the large X-effect and a specific role of 60 Mb–80 Mb interval of Chr X in reproductive isolation of Mmm×Mmd F1 hybrids fail to synapse properly their chromosomes at the pachytene stage of meiosis. Unsynapsed autosomes carry DMC1/RAD51 foci on unrepaired DSBs and are decorated by the phosphorylated form of histone H2AFX. The sex body containing X and Y chromosomes is often malformed or disappears, and transcriptional inactivation of sex chromosomes (MSCI) is disrupted trans- or cis-control. Mutations of various meiotic genes involved directly or indirectly in meiotic chromosome pairing and synapsis cause asynapsis of multiple autosomes in transPrdm9 gene on Mmd background causes male and female sterility associated with asynapsis and failure to form the sex body Mmm×Mmd hybrids Sycp1, Hormad1 or Mei4, the candidate genes regulating the Hstx2-controlled asymmetry of HS, also cause asynapsis. Contrary to genes acting in trans, certain structural mutations of chromosomes such as translocations or inversions can cause local, cis-acting asynapsis, followed by meiotic arrest and sterility trans- and cis- control of asynapsis in sterile Mmm×Mmd males, we modified the F1 hybrids using the intersubspecific chromosome substitution strains B6.PWD-Chr# in cis, because heterosubspecific chromosome pairs in male as well as in female meiosis were more prone to failure to synapse. The sensitivity of heterosubspecific chromosome pairs to asynapsis was strongly modified in trans by the Prdm9 and Hstx2 HS genes because only PWD/B6Prdm9 and PWDHstx2 allelic combinations on F1 hybrid background resulted in asynapsis of multiple autosomes in >90% of pachytene spermatocytes and full meiotic arrest. A few proteins required for meiotic chromosome alignment and pairing have been described cis-acting signals which ensure pairing and synapsis of homologous chromosomes are still unknown. Our assay of meiotic synapsis, comparing the consubspecific versus heterosubspecific pairing homologs, offers a genetic approach to solving the problem. Such experiments are in progress.More than 90% of primary spermatocytes of sterile Drosphila or mammals, and females in birds and Lepidoptera. Several hypotheses have been proposed to explain Haldane's rule, including the dominance theory, the faster-male theory, the faster X theory and meiotic drive Drosophila hybrid females homozygous for the recessive-acting X chromosome. In D. simulans×D. mauritiana or D. sechellia hybrids the female hybrids carrying two D. simulans Chr X were fertile, contrary to the prediction . We expect that analysis of recombinant heterosubspecific/consubspecific chromosome pairs could provide the genetic approach to identifying the cis-acting sites important for meiotic pairing and synapsis.The male-specific action of the The animals were maintained at the Institute of Molecular Genetics in Prague and Institute of Vertebrate Biology in Studenec (Academy of Sciences), Czech Republic. The project was approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Genetics, AS CR, protocol No. 141/2012. The principles of laboratory animal care  as well as specific Czech Law No. 246/1992 Sb. compatible with the corresponding EU regulations and standards, namely Council Directive 86/609/EEC and Appendix A of the Council of Europe Convention ETS123, were observed.M. m. domesticus) originated from The Jackson Laboratory . The PWD/Ph (PWD) and STUS strains were derived from wild M. m. musculusPWDad libitum access to a standard rodent diet  and acidified water. All males were sacrificed at the age of 60 to 70 days.C57BL/6J .All 124 male mice from (B6.PWD-Chr X×B6)F1×PWD cross and 71 male progeny from (B6.PWD-Chr X×B6)F1x B6 cross were genotyped using SSLP Chr X markers listed in Eight weeks old male progeny of the crosses were phenotyped for testes weight (TW), sperm count (SC) and percentage of abnormal spermatozoa as described scanone function. TW and logSC were modeled as continuous variables, fertility/sterility as a binary variable. Genotype probabilities between markers were calculated at a grid size of 5 cM and with genotyping error rate of 0.01%. Genome-wide significance was calculated by 1000 permutations and compared to α = 5% threshold.QTL mapping of recombinant males from (B6.PWD-Chr X×B6)F1×PWD and B6×(PWD×STUS) crosses was performed using the R 12.1 and its R/qtl package Three spermatogenic populations  were isolated using fluorescence-activated cell sorting (FACS) as described earlier http://www.bioconductor.org/) and the R project for statistical computing . The probes were annotated to Ensembl gene identifiers using the custom chip description file, which was based on NCBI build 37. The data were normalized using RMA (Affymetrix GeneChip Mouse Gene 1.0ST Array) and quantile normalization (Affymetrix GeneChip miRNA 1.0). We used Linear Models for Microarray Data Package, limma version 3.6 Total RNA was isolated from sorted cells and 14.5 dpc testis using the miRNeasy Mini isolation kit (QIAGEN) as recommended. RNA concentration was determined by NanoDrop (NanoDrop Technologies) and its integrity checked in Agilent 2100 bioanalyzer, RNA Lab-On-a-Chip (Agilent Technologies). The total RNA (20–30 ng for gene expression and 120 ng for miRNA expression) was converted to cRNA using the Affymetrix Two-Cycle Target Labeling kit according to the manufacturer's instructions or using the Affymetrix 3′ IVT Express Kit. Affymetrix GeneChip Mouse Genome 430.2.0 array, and Affymetrix GeneChip miRNA 1.0 Array was hybridized with cRNA. The data obtained from the experiments were analyzed using Bioconductor http://frodo.wi.mit.edu/). Sequences of primers are given in For qRT-PCR of protein-coding genes, reverse transcription of isolated RNA samples was carried out using Applied Biosystems (ABI) high-capacity cDNA reverse transcription kit. The quantification of mRNAs was performed using FastStart DNA Master SYBR Green I kit (Roche) and amplified in LightCycler 2000 (Roche). Reactions without reverse transcriptase were utilized as negative control. The assays were done in biological and technical triplicates. The data were analyzed using LightCycler Software version 3.5.3 (Roche). For validation of miRNA expression we used ABI TaqMan MicroRNA assays and followed the manufacturer's instructions. The reactions were cycled in Applied Biosystems 7300 Real-time PCR system, and associated software was used for data analysis. The reactions were also carried out using biological and technical triplicates and proper negative controls. The highest and stably expressed miRNAs, U6 non-coding RNA for sorted cells and Mir152 for 14.5 dpc testis, were used as the reference for data normalization. The primers were designed using Primer 3 software (http://samtools.sourceforge.net/SAM1.pdf) and deposited at the Sequence Read Archive (SRA) accession SRR942524.Exome sequence analysis was carried out for PWD/Ph mice at BGI Europe using Illumina HiSeq 2000 sequencers. The PWD exome sequence was aligned to NCBIm37 genome  and PWD BACs (for miRNAs) http://phenome.jax.org/). The dN∶dS ratio (an indicator of evolutionary selective pressure on genetic processes) of different X-linked protein-coding genes between rat and mouse was calculated using Ensemble Biomart.All the non-synonymous mutations between PWD and B6 for 4.7 Mb The meiocyte spreads were prepared by using the hypotonic protocol as described earlier http://www.graphpad.com/scientific-software/prism/#1).Multiple biological replicates of each genotype were analyzed for cellular phenotypes and RNA expression. The significance of body weight, testes weight, sperm morphology and breeding phenotypes was computed using Welsch's t-test. Sizes of chromatin areas covered by the hybridization signal in synapsed and unsynapsed autosomes were compared by Analysis of Variance (ANOVA) with Tukey's correction for multiple testing. Differences between cellular phenotypes were determined with χ2 test. All computations were done using R 2.15.0 or Graphpad Prism  and DXMit143 (69.28 Mb), indicating the position of the Hstx1 gene.Mapping of (EPS)Click here for additional data file.Figure S2Hstx2/Hstx1 critical region on Chr X. Predicted genes and pseudogenes are not included in this map. See text for details.Gene map of the (EPS)Click here for additional data file.Figure S3Hstx1/Hstx2 critical region. (A) Log fold-change of expression ratio of PWD versus B6 protein-coding genes in flow sorted testicular cells. Data from Affymetrix GeneChip Mouse Gene 1.0ST Array do not show any significant differences between both strains in expression levels in primary spermatocytes. Abbreviations: Z-EP – Zygotene Early Pachytene, M-LP - Mid-Late Pachytene, ST – Spermatids. (B) qRT PCR of expression levels of six candidate genes in testes of 14.5 d reciprocal F1 hybrid males (PWD×B6)F1 and (B6×PWD(F1). None of the differences is significant.Expression profiling of protein-coding genes within (EPS)Click here for additional data file.Figure S4Hstx2 on spermatogenic differentiation and synapsis of meiotic chromosomes. (A) Histological crossections of spermatogenic tubules of B6 control and (B6.PWD-Chr X.1×PWD)F1, abbreviated here DX.1PF1, and (B6.PWD-Chr X.1s×PWD)F1, abbreviated DX.1sPF1 hybrids. Note the vacuolar degeneration, heteropycnotic cells and absence of sperm cells in DX.1sPF1 hybrids. (B) Unsynapsed autosomes and sex chromosomes visualized by HORMAD2 and SYCP3 immunostaining. Early pachynemas with high and low frequency of autosomal univalents are shown. (C) Frequency of primary spermatocytes at various stages of meiotic progress in males of different genotype. (PWD×B6)F1, abbreviated PB6F1, and B6.PWD-Chr X.1s×PWD)F1 (abbreviated DX.1sPF1) sterile males display the same profile of primary spermatocyte stages.  Frequency of pachynemas with asynapsis and number of unsynapsed autosomes per cell are similar in PB6F1 and DX.1sPF1 sterile males. In DX.1PF1 males asynapsis occurs with lower frequency and the pachynemas with asynapsis have lower number of univalents.The effect of (EPS)Click here for additional data file.Figure S5Mmm and Mmd inbred strains. (A) Over 90% of pachytene spermatocytes in all three strain combinations ((PWD×B6)F1 – abbreviated PB6F1, (STUS×B6)F1 – STB6F1 and (PWD×SCHEST)F1 - PSCHF1) carry one or more asynapsed chromosome pair. Frequency of asynapsis of four analyzed autosomes varied between genotypes. Average number of univalents per cell was highest in PSCHF1 and lowest in PB6F1. (B) Distribution of pachynemas with a given number of unsynapsed chromosome pairs. Asynapsis varies widely within each genotype. A significant excess of cells with one unsynapsed pair occurs in B6PF1 hybrids.Pachytene asynapsis in sterile F1 hybrid males prepared by different combinations of (EPS)Click here for additional data file.Table S1Coordinates of the introgressed PWD sequence in B6.PWD-Chr X subconsomics (GRCmm38).(DOCX)Click here for additional data file.Table S2Meiotic stages of adult testes of B6.PWD-Chr.X# and PWD hybrids.(DOCX)Click here for additional data file.Table S3Testes weight and sperm count in male progeny of B6×(STUS×PWD), B6×(PWD×STUS) and reciprocal crosses (STUS×PWD)×B6, and (PWD×STUS)×B6.(XLSX)Click here for additional data file.Table S4PWD-independent HS QTLs.Intervals and candidate genes of Chr X(DOCX)Click here for additional data file.Table S5Hstx1/Hstx2 loci.Fertility of parental and F1 hybrid females with different allelic combinations at (DOCX)Click here for additional data file.Table S6Primers.(XLS)Click here for additional data file.


The axis passes through the mid-point of the 1,10 and 5,6 bonds of the N-heterocycle and through the Sn atom. The Sn atoms show a slightly distorted SnC2N4 octahedral coordination.In the asymmetric unit of the title compound, [Sn(C DOI: 10.1107/S1600536811001292/bt5460Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are members of the purinergic receptor family that are expressed in several cell types including neurons. A high concentration of ATP is required for the channel opening of P2X7Rs compared to other members of this receptor family. Recent work suggests that ATP binding to members of the P2X receptor family determines the diffusion and localization of these receptors on the plasma membrane of neurons. Here, we employed single particle tracking photoactivated localization microscopy (sptPALM) to study the diffusion and ATP-dependence of rat P2X7Rs. Dendra2-tagged P2X7Rs were transfected in hippocampal neurons and imaged on proximal dendrites. Our results suggest the presence of two populations of P2X7Rs within the extra-synaptic membrane: a population composed of rapidly diffusing receptors and one stabilized within nanoclusters (~100 nm diameter). P2X7R trajectories were rarely observed at synaptic sites. P2X7R mutations in the ATP-binding site (K64A) or the conserved phosphorylation site (K17A) resulted in faster- and slower-diffusing receptors, respectively. Furthermore, ATP differentially accelerated wild type and K17A-mutant receptors but not K64A-mutant receptors. Our results indicate that receptor conformation plays a critical role in regulating ATP-mediated changes in P2X7R diffusion and micro-organization. In the hippocampus of EGFP-P2X7R expressing mice, expression can be seen primarily in the dentate gyrus and CA3 region (Cornu Ammonis) suggesting a cell-type specific expression .ATP-gated purinergic P2X receptors (P2XRs) form homo- or hetero-trimeric receptors that can be composed of 7 different subunits, P2X1-P2X7. Slow-desensitizing P2X7Rs are unique members of the ATP-gated P2X receptor family with a characteristic long C-terminus (279 amino acids) and display unusually high ECThe development of quantum dot based SPT (QD-SPT) studies has changed our understanding of activity-dependent dynamics of neurotransmitter receptors  and generated as described previously . The EGFP sequence was downstream of P2X7 coding region between 5 cells/well on 18 mm coverslips pre-coated with 80 μg/ml poly-D, L-ornithine (Sigma). Freshly dissociated cells were plated in neuronal attachment media consisting of 10% horse serum (PAA Labs), 1 mM sodium pyruvate (Life Technologies) and 2 mM glutamine (Life Technologies) in MEM (Life Technologies). Three hours after plating, media was replaced with neurobasal media containing 2 mM glutamine and 2% B27. Cells were maintained by replacing one-fourth of the medium with fresh culture medium every week. Transfection was performed using Lipofectamine-2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were transfected with 0.4 μg plasmid per coverslip at days in vitro (DIV) 12. Imaging was performed on DIV 14.Primary hippocampal cultures were prepared from 18-days-old Sprague–Dawley rat embryos. Neurons were plated at a density of 0.6 × 10Immunocytochemistry was performed following fixation of cells in 4% (w/v) paraformaldehyde  and permeabilization with 0.1% triton-X. Cells were incubated with primary antibodies against TAU (1:1000), MAP2 (1:1000) or Synapsin (1:800) for 1 h at room temperature. After washing, cells were incubated for 45 min at room temperature with appropriate secondary antibody and mounted on slides with Vectashield (Vector Labs).The PALM setup and imaging conditions used have recently been described in detail  to correct drift. Imaging was performed for 20,000 frames at 20 Hz. For sptPALM experiments, beads were not used and imaging was performed for 5000–6000 frames at 50 Hz. Imaging was performed in MEM medium without phenol red (Invitrogen) containing 2% B-27, 2 mM glutamine, 1 mM pyruvate, 33 mM glucose, and 20 mM HEPES.Multi-Trace Tracking (MTT) algorithm was used without the tracking feature  = 4D2−5t + 4σ2x, with σx as the spot localization accuracy in one direction. Area explored is defined as the total surface area covered by the trajectory divided by the number of steps in the trajectory.Tracking of PALM traces and diffusion calculation was based on the mean square displacement (MSD) approach  and experiments performed at DIV 14. To visualize the distribution and localization of P2X7-Dendra2 receptors, we performed immunocytochemistry to label axons (tau), dendrites (MAP2), and synapses (synapsin). P2X7-Dendra2 receptors showed a ubiquitous and uniformly diffused expression in most of the transfected neurons  Figure . Two repNotably, the majority of P2X7-Dendra2 receptor detections were not clustered, suggesting a predominant presence as single molecules. The observed nanoclusters appear to be enriched in some dendrites. Altogether, these results suggest that transfected P2X7Rs exist in two populations, one as single molecules and a second stabilized in nanoclusters.2/s) are: extra-synaptic = 0.077 (n = 1246); synaptic = 0.054 (n = 59); free = 0.090 (n = 597); trapped = 0.038 (n = 327).Dendra2 undergoes a green-to-red shift in emission wavelength (photo-conversion) when exposed to UV light (405 nm laser). Following photo-conversion, Dendra2 remains switched on for a short time period before irreversibly photobleaching . Mutation of lysine at position 17 removes the putative protein kinase-C (PKC) phosphorylation site (15TXK17) without altering receptor gating  ATP was added to the recording medium prior to imaging. Changes in diffusion coefficient were then measured for free and trapped populations. ATP-treated cells showed a small but significant increase in the diffusion coefficient of the free population of WT-P2X7Rs Figure , Table 100 μM ATPWT = 0.2175 (n = 2384), WT + ATP = 0.1902 (n = 2457), K17A = 0.1305 (n = 816), K17A + ATP = 0.1104 (n = 556), K64A = 0.2387 (n = 2911), K64A + ATP = 0.1923 (n = 2225). The above data was obtained from trajectories with a minimum length composed of 5 detections. A similar reduction in area explored was observed with a minimum of 10-point trajectories. These observations suggest that receptors “trapped” within nanoclusters exhibit increased confinement in the presence of extracellular ATP. Notably, ATP had the highest effect on K64A-mutant receptors, which exhibit a disruption in the ATP-binding site  of P2X7R trajectories that were trapped in nanoclusters. This parameter is independent of the length of the trajectories and gives a good estimate of receptor confinement. We observed a reduction in the area explored/length for both WT and mutant receptors Figure . The med05 n = 81, K17A + Previous studies using QD based SPT suggested that members of the P2XR family are not enriched at synapses , P2XR-interacting proteins  interacting molecules, which may induce clustering at non-synaptic localizations. In fact, we observed non-synaptic clusters of P2X2R-subtype in spinal cord neurons supporting the existence of such receptor-stabilizing nanoclusters , resulting in over-spill of single P2X7Rs. Compared to WT-P2X7Rs, the diffusion of P2X7Rs lacking the putative PKC phosphorylation site (K17A-mutated) was more confined. Phosphorylation-dependent change in receptor conformation could be a possible explanation for the slower diffusion of K17A-mutant. However, recent work argues against this possibility. It has been reported that mutation of threonine within the conserved PKC site (15TXK17) resulted in a more sensitive agonist response. Contrarily, mutation of K-17 did not alter receptor gating, even though the phosphorylation site was removed  of the ATP-binding pocket also affect the diffusion coefficient of P2X7Rs, exogenous ATP was added to the imaging media. Following addition of ATP, an increase in the mobility was observed for WT- and K17A- but not for K64A-P2X7Rs. These results further support that perturbation of the ATP-binding pocket through either a point mutation (K64A) or through the opening of receptors after addition of ATP, causes a change in the diffusion coefficient of P2X7Rs. Consequently, ATP-dependent P2X7R conformation regulates its diffusion. It is likely that structural rearrangement within the extracellular ATP-binding pocket leads to changes in conformation of intracellular N- and/or C-terminus, thus modifying diffusion behavior. It should be emphasized that several other factors may contribute to the observed changes in diffusion such as basal phosphorylation state, interaction with scaffold, maturation of neurons, cell-type, network activity, etc. More work is needed to further understand the mechanism of such regulation.ATP also had an effect on trapped receptors. Both WT and K64A-P2X7Rs trapped in nanoclusters exhibited a decrease in the area explored following ATP treatment. Thus ATP indirectly stabilizes P2X7Rs present within nanoclusters. Indeed, more work is needed to identify the mechanism, but involvement of other endogenous P2XR subtypes cannot be ruled out. ATP-dependent regulation of P2XR mobility and clustering has been demonstrated by several studies  adenosine 5′-triphosphate) that binds the same site as ATP was found to increase clustering of P2X2Rs (Shrivastava et al., Amulya N. Shrivastava, Pamela C. Rodriguez, Marianne Renner, and Antoine Triller designed the experiments and wrote the manuscript. Amulya N. Shrivastava and Pamela C. Rodriguez performed the experiments. Marianne Renner developed the tools for analysis of sptPALM data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5pV326N cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5pWT cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.Cell adhesion molecules in  Candida albicans is a human commensal fungus that is pathogenic when its growth becomes uncontrolled, especially in imunocompromised individuals. Under such conditions these eukaryotes can form biofilms that are resistant to a variety of environmental assaults, including antimicrobials C. albicans to various substrates and each other. Among many adhesins, members of the Als family of glycoproteins are particularly active in cell aggregation, adhesion to endothelia and epithelia, formation of biofilms and pathogenesis in mouse models. There are eight ALS gene loci, with high heterozygosity, so Als adhesin sequences and binding specificities are diverse. However, all Als proteins have similar modularity and domain structure  Previously, we showed that the amyloid sequence in the T region of Als proteins mediates amyloid formation, and that amyloid binding dyes can inhibit aggregation in the S. cerevisiae cells expressing Als5p form large aggregates, similar to those seen in C. albicansS. cerevisiae (WT) and the substitution sequence (Als5pV326N) were expressed on the yeast cell surface, with non-amyloid Als5pV326N expression slightly greater than Als5pWT . In contrast the small aggregates of cells expressing Als5pV326N showed little fluorescence under these conditions . This concentration had no visual effect on the ability of the Als5pV326N to mediate formation of small aggregates (vs. 3L). Furthermore, this concentration is at least a thousand-fold lower than the concentrations that inhibit growth and fungal cell wall biogenesis WT and Als5pV326N cells to bind to the beads and form aggregates  and S. cerevisiae expressing Als5pWT (vs. 3I and 3J). Thioflavin T had no effect on the smaller aggregates of S. cerevisiae Als5pV326N cells (Thioflavin T (5 µM and 450 µM) also decreased cell-cell adhesion in 6N cells  or on em6N cells . These d1-431, including the Ig-like and Thr-rich amyloid region; and Als5p1-664, which also includes the tandem repeat region in vitro binding to fibronectin and polystyrene, the results were similar to those for the wild type Als5 protein within experimental error for both the 431-residue and 664-residue fragments.  rapidly forms insoluble amyloids V326N by providing a stable amyloid template. S. cerevisiae expressing either form of Als5p and C. albicans were incubated without (S. cerevisiae expressing Als5pV326N (vs. 4J). The peptide had no detectable effect on the aggregation of empty vector, or Als5pWT-expressing S. cerevisiae or on C. albicans strains . A scrambled-sequence peptide with the same amino acid composition as the Als5p amyloid-forming sequence (VITGVTNIRTSVA) did not induce aggregation, indicating that the observed effect was specific to the wild type sequence (A tridecapeptide consisting of the sequence of Als5p without  or with  without  during ts5pV326N vs. 4J. sequence .vs. 4M–4P). The small Als5pV326N aggregates lacked intense thioflavin T fluorescence . Thus, exogenous amyloid-forming homologous sequence peptide induced increased aggregation in non-amyloid cells, and increased amyloid fluorescence in aggregating cells as well.To determine if the peptide-induced adhesion was accompanied by formation of amyloid-like interactions, cells were stained with thioflavin T  during the bead assay . The few remaining aggregates looked similar to those observed with Als5pV326N (V326N strains (vs. 4Q and 4B vs. 4R). A scrambled V326N peptide (VITGNTNIRTSVA) did not block aggregation indicating that the inhibitory effect is specific to the V326N sequence . The peptide had no effect on the fluorescence of the empty vector and Als5pV326N aggregates .We have tested this hypothesis by a detailed determination of phenotype for single site substitution V326N in the 1419 amino-acid Als5p sequence. This mutation is in the seven residue amyloid-forming sequence Iin vitro and also altered the activity of cell-bound Als5p. The mutation did not significantly affect cell wall localization of the protein  and was grown in YPED with 80 mg/L uridine at 30°C. S. cerevisiae strain W303-1B  was grown in CSM with galactose at 24°C. Cells harboring empty vector (pJL1-EV), or expressing Als5pWT or Als5pV326N were grown in CSM with galactose lacking Trp.WT was generated by restriction digestion of pGK114 with BamHI and XhoI which released the Als5p sequence from the vector backbone -BamHI and 3′-NotI restriction sites NotI site and a 3′XhoI site was generated by PCR and was ligated to the modified vector to make pJL1. The resulting construct was verified by sequencing .V5-tagged Als5pV326N was generated by digestion of pGK114 with SphI and AleI to generate a 363bp fragment at nucleotide position 1242 to 1605, containing the target sequence to be mutated. This fragment was subcloned into pGEM-T vector and mutagenized using Quickchange  with mutagenic primer 5′-GAA TAG TGA TGC CGG ATC TAA CGG TAT TAA CAT TGT TGC TAC AAC TAG AAC AGT TAC AGA CAG-3′. The correct mutation was verified by sequencing. The mutated fragment was released from the vector with the same enzymes used in its generation, and placed into the corresponding position of pJL1. The resulting product, pJL1V326N was verified by sequencing to determine the presence of the full-length Als5pV326N.Als5pBamHI and XhoI and ligating in the multiple cloning site from p414 . The pJL plasmids were transformed into S. cerevisiae strain W303-1B.pJL-EV was produced by restriction digestion of pJL1 with 6, were added to 1×108 cells in 13×100 mm glass test tubes and the suspension was shaken at 200 rpm at 24°C 30-45 minutes. Adherent cells were separated and washed over a magnet, resuspended in TE buffer and observed by microscopy. Thioflavin T fluorescence was excited at 425 nm and monitored at 510 nm.Cell aggregation was observed using a previously described method with minor modifications 7 cells/ml. Cell suspension, 100 µl/well, was added to a polystyrene non-tissue culture plate with 96 wells. Each sample was done in triplicate. The cells adhered to the surface for 1.5 hours at room temperature. The polystyrene surface was washed with TE, sterile liquid media was added and the adherent cells were incubated overnight at 30°C. The next day the non-adherent cells were washed away with TE. Adherent cells were stained with crystal violet and observed by microscopy and imaged. Adhesion was quantified by solubilization of the crystal violet in 10% SDS for 1.5 hours and measuring the absorbance at 570 nm.Adhesion to polystyrene was assayed using a protocol previously described 3N4 cantilevers . Cells were immobilized by mechanical trapping into porous polycarbonate membranes (Millipore), with a pore size similar to the cell size. After filtering a concentrated cell suspension, the filter was gently rinsed with buffer, carefully cut (1 cm ×1 cm), attached to a steel sample puck (Veeco Metrology Group) and the mounted sample was transferred into the AFM liquid cell while avoiding dewetting. The spring constants of the cantilevers were measured using the thermal noise method , yielding values ranging from 0.008 to 0.021 N/m. All force measurements were recorded with a loading rate of 10,000 pN/s. AFM tips were functionalized with anti-V5 antibodies or Als5p1-431 protein as described earlier AFM measurements were performed at room temperature (20°C) in buffered solutions , using a Nanoscope IV Multimode AFM  and oxide sharpened microfabricated SiFigure S1V326N substitution proteins.ELISA assays of binding of Als5p The upper graph shows binding of increasing concentrations of proteins to different concentrations of fibronectin. The lower graph denotes binding of the proteins to polystyrene. The constructs shown are Ig-T-TR, wildtype (•) and V326N (▾), and Ig-T wildtype (○) and V326N (Δ). The protein concentration is 2.3±0.7μM for the fibronectin binding. The assays were carried out as previously described (TIF)Click here for additional data file.Figure S21-664 protein.Far UV Circular Dichroism spectra of Als5p  Wildtype protein is represented by the solid line and the V326N substation by the dashed line at 20°C.(TIF)Click here for additional data file.Figure S3Effects of scrambled V326N (VITGVTNIRTSVA) and wild type peptide (VITGNTNIRTSVA) on cellular aggregation.S. cerevisiae expressing no Als5p (EV), Als5pV326N, Als5pWT, or C. albicans were aggregated in the absence and presence of 2µg/ml scrambled wild type (S-wild type) or 200 µg/ml scrambled peptide (S-V326N). The diameter of the beads is 2.8 µm, and all images are at the same magnification.(TIF)Click here for additional data file.Figure S4Amyloid binding dye Congo red reduces binding and aggregation on polystyrene biofilms.C. albicans or S. cerevisiae expressing Als5pWT, Als5pV326N, or no Als5p (EV) adhered to a polystyrene surface in the absence and presence of 100 nM or 300 µM Congo red for 1.5 h. Adherent cells were grown overnight, stained with 1% crystal violet and imaged.(TIF)Click here for additional data file.Figure S5Detection and unfolding of single Als proteins in C. albicans. (A) Principle of the single-molecule detection experiment. C. albicans cells are probed, in buffer, using AFM tips derivatized with Als5p1-431. (B) Force extension curves obtained by stretching ALS proteins showed periodic features reflecting the sequential unfolding of the TR domains. (C) Plot of the rupture distances as a function of the number of unfolded TR.(TIF)Click here for additional data file.