The survey of expression patterns, biochemical characters, and intracellular localizations of cystatins in tobacco reveals their widespread roles in gamete development, embryogenesis, and seed formation. Nicotiana tabacum were identified using an expressed sequence tag (EST)-based gene clone strategy. Analysis of their biochemical properties showed that nine of them have the potency to inhibit the activities of both commercial cathepsin L-like proteases and extracted cysteine proteases from seeds, but with different Ki values depending on the types of proteases and the developmental stages of the seed tested. This suggests that cystatin-dependent cathepsin L-like proteolytic pathways are probably important for early seed development. Comprehensive expression profile analysis revealed that cystatin family genes showed manifold variations in their transcription levels in different plant cell types, including the sperm, egg, and zygote, especially in the embryo and seed at different developmental stages. More interestingly, intracellular localization analysis of each cystatin revealed that most members of cystatin families are recognized as secretory proteins with signal peptides that direct them to the endoplasmic reticulum. These results suggest their widespread roles in cell fate determination and cell–cell communication in the process of sexual reproduction, especially in gamete and embryo development, as well as in seed formation.Cystatins are tightly bound and reversible inhibitors of cysteine proteases in C1A and C13 peptidase families, which have been identified in several species and shown to function in vegetative development and response to biotic/abiotic stresses in plants. Recent work revealed their critical role in regulating programmed cell death during embryogenesis in tobacco and suggested their more comprehensive roles in the process of sexual plant reproduction, although little is known about cystatin family genes in the processes. Here, 10 cystatin family genes in  Oryza sativa, Arabidopsis thaliana, and Populus trichocarpa  analysis of each contig was performed using OMEGA, and the BLASTP program of the National Center for Biotechnology Information (NCBI) with intact or partial deduced protein sequences of each contig. The contigs with a partial or intact cystatin domain based on information obtained were selected as candidates for further study.A total of 3.5×10Supplementary Table S1 available at JXB online). RT–PCR was carried out in a 50 μl PCR mixture containing 5 μl of 10× Ex Taq buffer, 2.5mM MgCl2, 200 μM dNTPs, 0.2 μM of primers, 1.2U of Ex Taq DNA polymerase (Takara), and cDNA prepared from different tissues. Conditions for PCR on the T100™ Thermal Cycle PCR system (Bio-Rad) are as follows: initial denaturation at 94 °C for 2min; 35 amplification cycles with denaturation at 94 °C for 30 s, annealing at Tm –5 °C for 30 s; extension at 72 °C for 1min; and a final incubation at 72 °C for 5min.After ORF analysis, full-length cDNA of each contig was obtained through the rapid amplification of cDNA ends (RACE) approach. Full-length sequences were confirmed by reverse transcription–PCR (RT–PCR) with specific primers at the 5’ and 3’ end, respectively , and cDNA prepared from different tissues. Conditions for RT–qPCR were as follows: activation of FastStart Taq DNA polymerase at 95 °C for 10min, and >40 cycles (95 °C for 15 s and 60 °C for 1min) with a Rotor-Gene 6000 system (Corbett Research). The data analysis was conducted according to a previous procedure , and total RNAs of seeds at different stages were extracted with RNAqueous™ (Ambion). All total RNAs were treated with RNase-free DNase I (Promega) and cDNAs were synthesized using ReverTra Ace (Toyobo) under the conditions recommended by the manufacturer. mRNA isolation from sperm cells, egg cells, zygotes, apical cells, basal cells, and embryos at different stages and cDNA synthesis were performed according to a previous procedure (Supplementary Table S2 at JXB online) and inserted into the pMXB-10 vector (NEB). The resulting plasmids were transformed into Escherichia coli BL21 (DE3) (Novagen). The recombinant cystatins were expressed and purified according to the manufacturer’s instructions. The purified cystatins were re-purified by ion exchange chromatography with a Bio-Scale™ Mini UNOsphere™ Cartridge Q/S or a Bio-Scale™ Mini CHT Type I Cartridge (Bio-Rad) on BioLogic DuoFlow™ system (Bio-Rad). The final protein concentrations were quantified using a Coomassie Plus kit (Thermo) with bovine serum albumin as the standard.The coding regions of cystatin family genes lacking the stop codon and signal peptide sequences were cloned , human liver cathepsin L (Sigma-Aldrich), cathepsin B (Sigma-Aldrich), and cathepsin H (Merck), substrate hydrolysis progress curves of each cysteine protease were monitored according to a previous method , and the supernatants were collected for further analysis. The final concentrations of total protein in the supernatants were quantified using a Coomassie Plus Kit (Thermo) with bovine serum albumin as the standard.For the determination of the inhibitory potency of each cystatin against total extracts from seeds at different stages, all samples were frozen in liquid nitrogen and ground in a mortar using a pestle. After grinding, the samples of seeds were suspended in 50mM MES (pH 6.0), 2mM EDTA, 10% glycerol, 0.1% CHAPS, 0.01% Brij-35, 2% polyvinylpolypyrrolidone (PVPP), 10mM N-carbobenzoxyloxy-Phe-Arg-7-amido-4-methylcoumarin), Z-RR-AMC (N-carbobenzoxyloxy-Arg-Arg-7-amido-4-methylcoumarin), and Bz-FVR-AMC  susceptible to degradation by cathepsin L-, B-, and H-like proteases, respectively. Hydrolysis was allowed to proceed at 30 °C in a 100 μl assay mixture containing 50mM sodium phosphate (pH 6.0), 5 μg of total protein, 25 μΜ substrate, 10mM l-cysteine, 1mM EDTA, and 0.01% Brij-35 with or without the addition of 1 μΜ recombinant cystatins . The activity levels were monitored using a Spectra Max M2 (Molecular Device Co.) with excitation and emission filters of 360nm and 455nm, respectively.The inhibitory potency of each recombinant cystatin against total protein from tobacco seeds was tested by monitoring hydrolysis of the substrates Z-FR-AMC (35S::eGFP-NOS was firstly constructed in pRS300 to generate the pRS300-35S-eGFP-NOS vector. The full-length cystatin coding sequences (without stop codons) were amplified and inserted in-frame with enhanced green fluorescent protein (eGFP) into the vector pRS300-35S-eGFP-NOS to generate 35S::NtCYS-eGFP-NOS expression vectors (Supplementary Table S2 at JXB online). 35S::NtCYS-eGFP-NOS expression vectors were co-expressed with an endoplasmic reticulum (ER) marker containing an N-terminal signal peptide derived from a vacuolar basic chitinase of A. thaliana and the C-terminal amino acid sequence HDEL (RFP-ER) . Coating by gold particles and bombardment were performed according to the manufacturer’s instructions (Bio-Rad Laboratories). Transformed A. cepa epidermis was observed under a confocal microscope (Olympus FluoView FV1000). Images were processed with Adobe Photoshop.For intracellular localization analysis of each cystatin identified in tobacco, 5 EST sequences from tobacco were downloaded from the database at the NCBI, and constructed into a local BLAST database. An tBLASTn search was carried out using conserved protein sequences of the cystatin family, and an expectation value <1.0×10–5 was considered to indicate true cystatin family genes. A total of 119 EST sequences related to cystatin family genes were obtained and assembled into 15 contigs, and redundant sequences were omitted manually. The full length of these sequences was obtained through the RACE technique, and detailed information on each gene is given in N. tabacum, including a known cystatin NtCYS1. Thus, the others genes were designated in numerical sequence.To identify cystatin family genes in tobacco, 3.5×10GAPDH  and 35 cycles for cystatin genes. The transcripts of nine novel predicted cystatin genes can be detected in different tissues of tobacco as shown in NtCYS8 is only detected in early seeds, but not in the other tissues tested, indicating its specific roles in early seed development. These data suggest that all predicted novel cystatin genes exist in tobacco, and display different expression patterns, implying their specific roles in different stages of tobacco development.To confirm further the existence of the predicted cystatin genes in tobacco, cDNA prepared from different tissues including leaf, stem, root, pollen, anther, and seeds at stages 1, 5, and 9 were selected as templates for RT–PCR. PCR parameters were optimized to identify cystatin genes in these tissues: 28 cycles for the housekeeping gene N. tabacum. The sequence features and functional motifs of each of the cystatins were then investigated. Among them, nine cystatins have a molecular mass in the range of 10.8–16.1kDa, and only NtCYS3 has a molecular mass of ~28.1kDa with a C-terminal extension. Prediction of the signal peptide using SignalP 4.0 : (i) one or two glycines at the N-terminus are conserved (except in NtCYS8); (ii) a ‘LARFAV’ motif is present with related substitutions in all proteins (except in NtCYS10); (iii) the active site ‘QxVxG’ is essentially conserved, although an additional amino acid was found in NtCYS8 (‘QVVATG’); (iv) a tryptophan is conserved in the C-terminus of most cystatins, with the exception of NtCYS1 and NtCYS8; (v) two novel motifs, ‘VWxKPW’ and ‘KxLxxF’, were found in the C-terminus of all cystatins with related substitutions; and (vi) a C-terminal extension with ‘SNSL’ was detected in NtCYS3, which is a putative site for the inhibition of the activities of legumain-like proteases.As described above, 10 cystatin family genes including nine novel genes have been identified in nalP 4.0 , indicatSupplementary Fig. S2 at JXB online). The ‘QxVxG’ reactive site is located in the loop between the second and third β-strand of most cystatins, with the exceptions of NtCYS3, NtCYS4, and NtCYS10. The three-dimensional structure of the extended C-terminus of NtCYS3 is different from that of other typical cystatins, with two α-helices and 12 β-strands, which suggests that the changes in three-dimensional structure may contribute to their specific biochemical properties.To compare the similarities and differences of the structures of these cystatin proteins in tobacco, the secondary structures of cystatins were predicated using PSIPRED v3.3  . Group BE. coli using the IMPACT™ expression system (NEB) and purified in a soluble form. Recombinant proteins of the expected size, free of protein contaminants, were obtained through affinity purification and ion exchange chromatography  followed by cathepsin H . NtCYS4 is the strongest inhibitor for cathepsin L with Ki values of 3.4×10–12 M. NtCYS5 is the strongest inhibitor for papain and cathepsin H, with Ki values of 9.3×10–12 M and 5.1×10–11 M, respectively, whereas NtCYS10 is the strongest inhibitor for cathepsin B with Ki values of 6.4×10–9 M. NtCYS8 with the non-typical reactive site ‘QVVATG’ is a putative inhibitor with no visible inhibitory potency for all the cysteine proteases tested, suggesting that the typical reactive site ‘QxVxG’ may play important roles in their biochemical function.Most cystatins can inhibit the activities of cysteine proteases in the peptidase C1A family , suggesting that it may play important roles in sperm development. Cystatins in egg cells usually display a relatively lower level compared with those in sperm cells or zygotes, except for NtCYS3 and NtCYS7. In contrast, most of the cystatins are abundant in zygotes compared with sperm cells and egg cells, with the exceptions of NtCYS5 and NtCYS7. In addition, NtCYS4, NtCYS9, and NtCYS10 show a significantly higher expression level in the zygote (>2-fold more than the sperm cell and egg cell). These results indicate that gamete-specific or preferential expression of cystatins exists in tobacco, and their differential expression probably plays distinct roles in gamete development and zygote formation.The analysis showed that most of the tobacco cystatin genes can be detected in sperm, egg cells, and zygotes . HoweverNtCYS1, which controls the onset of suspensor PCD by directly regulating the activity of the cathepsin H-like protease NtCP14, has been identified  showed significantly higher expression in basal cells (>2-fold), indicating that other cystatin family genes excluding NtCYS1 may also function in basal cell differentiation and development.The first asymmetric division of the one-celled proembryo (late zygote) is the beginning of sporophytic development in the plant, and usually gives rise to two daughter cells with distinct developmental fates. A small apical cell is the founder of a cell lineage generating the embryo proper, whereas a larger, basal cell establishes a cell lineage leading to the suspensor, which connects the embryo proper to maternal tissues . The second expression peak was found after fertilization and during proembryo formation (seeds at 4–8 d after pollination). NtCYS2, NtCYS4, NtCYS8, NtCYS9, and NtCYS10 showed peak expression during this process. The last expression peak was found at the stage of organ expansion and embryo maturation. The majority of cystatins except NtCYS8 showed another expression peak at this stage.Flowering plant seeds build a highly elaborate functional unit with the aim of propagating offspring, which usually consists of integuments, embryo, and endosperm. From a descriptive point of view, plant seed formation can be divided into three major stages in which three distinct developmental and physiological events occur. The first stage is from immediately after fertilization to proembryo formation, the second is embryo transition, and the last is organ expansion and embryo maturation  reach a peak when anthers develop into stage 3, and decrease gradually at later stages. Cystatin and their targets are known to be associated with various types of PCD . The exact roles of NtCYS2 in pollen development remain to be elucidated in a further study.Another striking feature of the expression profile of cystatin family genes is that several of them are abundant in male reproductive organs, especially in the anther at different stages , 9. TheySupplementary Table S3 at JXB online). Each recombinant cystatin showed a signiﬁcantly reduction (by ~90.8–98%) in the cathepsin L-like activities present in tobacco seeds at stages 1–6, and cathepsin H-like activities were also inhibited by some cystatins (by ~76.7–41.5%), whereas only a few cystatins can inhibit cathepsin B-like activities (by ~13.3–42.4%). In contrast, when embryos develop into stage 7, the inhibitory potency of each cystatin against cathepsin L-like activities decreased significantly (by ~14.3–29.7%). Similar to the inhibitory potency against cathepsin L-like activities, the inhibitory potency of each cystatin against cathepsin B- and H-like activity also decreased significantly. All these data suggest that cystatin-dependent cathepsin L-like proteolytic pathways are important for early seed development.The expression profile analysis shows that the transcripts of most cystatin genes in tobacco can be detected in seeds at different stages and their transcription levels show dynamic changes during the whole process of seed development, suggesting that these members may have potential roles in seed development. To confirm the potential function of the proteolytic pathway of cystatin-dependent cysteine proteases in seed development, the inhibitory capabilities of each recombinant cystatin against different types of cysteine proteases in tobacco seeds were tested. Total proteins were extracted from seeds at stage 1, 3, 6, 7, and 9, respectively. Each cystatin was then used to inhibit proteolytic activities in extracts from tobacco seeds at different stages, using substrates Z-FR-AMC (for cathepsin L-like proteases), Z-RR-AMC (for cathepsin B-like proteases), and Bz-FVR-AMC (for cathepsin H-like proteases), respectively. The assays were carried out by adding 1 μM of each cystatin to 5 μg of soluble protein extracts from seeds. The results indicated that each cystatin inhibited primarily the activities of cathepsin L-like proteases from early seeds at stages 1–6, and then the activities of cathepsin H-like proteases ; whereas two cystatins (NtCYS6 and NtCYS7) without signal peptides showed similar distributions to GFP alone. They could be detected in both the cytoplasm and nucleus. Surprisingly, two other cystatins (NtCYS2 and NtCYS3) with signal peptides were also targeted to the nucleus like HvCPI-1 and HvCPI-4 reported in H. vulgare  under the following accession numbers: NtCYS1 (KF113570), NtCYS2 (KJ725113), NtCYS3 (KJ725114), NtCYS4 (KJ725115), NtCYS5 (KJ725116), NtCYS6 (KJ725117), NtCYS7 (KJ725118), NtCYS8 (KJ725119), NtCYS9 (KJ725120), and NtCYS10 (KJ725121).Sequence data for the cystatin family genes in tobacco can be found in GenBank (JXB online.Supplementary data are available at Figure S1. Sequence alignment of cystatin protein sequences in tobacco.Figure S2. Predicated three-dimensional structures of cystatins in tobacco.Figure S3. Intracellular localization of tobacco cystatins in A. cepa epidermal cells.Table S1. Primers used in RT–PCR and RT–qPCR.Table S2. Primers used in vector construction.Table S3. Protease activities in extracts from developing tobacco seeds with or without recombinant cystatins.


Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable  and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit  was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine  tRNA molecules function as adapters in protein synthesis, bringing amino acids to the ribosome and reading the genetic code through codon-anticodon base pairing. When the tRNA contains a uridine residue in the “wobble position” of its anticodon, which base-pairs with purine residues in the third position of a cognate codon, it is almost always chemically modified and modification is required for efficient decoding. In eukaryotic cells, these wobble uridine modifications require a conserved protein complex called Elongator. Our work shows that Elp1, Elongator's largest subunit, is phosphorylated on several sites. By blocking phosphorylation at these positions using mutations, we identified four phosphorylation sites that are important for Elongator's role in tRNA modification. We have also shown that Hrr25 protein kinase, a member of the casein kinase I (CKI) family, is responsible for modification of two of the sites that are important for Elongator function. Phosphorylation appears to affect interaction of the Elongator complex both with its kinase (Hrr25) and with Kti12, an accessory protein previously implicated in Elongator function. Our studies imply that Elp1 phosphorylation plays a positive role in Elongator-mediated tRNA modification and raise the possibility that wobble uridine modification may be regulated, representing a potential translational control mechanism. IKBKAP gene encoding Elongator's largest subunit leads to embryonic lethality and the protein is crucial for vascular and neural development IKBKAP mutations, while mutations in other Elongator subunits have been associated with Amyotrophic Lateral Sclerosis Caenorhabditis elegans is also involved in neuronal function and development Elongator is a conserved, multi-subunit protein complex containing six different polypeptides (Elp1-Elp6), first discovered in yeast in association with the elongating form of RNA polymerase II and initially proposed to play a role in transcriptional elongation in vitroin vivoKluyveromyces lactis that kills other yeasts including Saccharomyces cerevisiaeWhile it is therefore clear that Elongator is important for neural function in higher organisms C. elegans5 (5-methoxycarbonylmethyl) and ncm5 (5-carbamoylmethyl) groups to uridine when present at the ‘wobble’ position (U34) in tRNA anticodons. Eleven out of 13 such tRNAs in yeast contain either mcm5U, ncm5U or 5-methoxycarbonymethyl-2-thiouridine (mcm5s2U) in the wobble position and addition of the mcm5 and ncm5 moieties requires Elongator Glu(UUC) by cleaving the anticodon on the 3′ side of U34, but the mcm5 modification is necessary for tRNA recognition and cleavage However, while these apparently diverse roles could imply that Elongator is multifunctional, work in yeast 5/ncm5 modifications are needed to confer full decoding competence on these tRNAs Lys(UUU) and tRNAGln(UUG), can suppress all the phenotypes associated with loss of Elongator function apart from zymocin resistance, which remains unaffected because elevated levels of the two tRNAs do not restore the tRNAGlu(UUC) modification required for cleavage by zymocin Lys(UUU), tRNAGln(UUG) and tRNAGlu(UUC) to the ribosomal A-site in vitro reaction containing SAM, acetyl-CoA, tRNA and Na2S2O4Wobble uridine-containing tRNAs read codons ending with a purine, and the mcmHRR25 (encoding a casein kinase I) or SIT4 (encoding a protein phosphatase) that conferred zymocin resistance hrr25 mutants and as a hyperphosphorylated isoform in sit4 mutants, whereas wild-type cells contained similar amounts of both isoforms Previously, we reported that the largest subunit of Elongator (Elp1) is a phosphoprotein and identified mutations in either 2, followed by tandem mass spectrometry to locate sites of phosphorylation. To maximize the chance of detecting phosphopeptides, in addition to isolating Elongator from wild-type yeast cells we also prepared and analyzed the complex from a sit4Δ strain, in which lack of Sit4 phosphatase leads to Elp1 hyperphosphorylation kti12Δ mutant in which Elongator is hypophosphorylated To identify sites of phosphorylation in Elongator we affinity isolated the complex from yeast cells expressing a TAP-tagged version of Elp1. Tryptic digests of the affinity-purified material were subjected to phosphopeptide enrichment involving a two-step procedure using Hypersep SCX and TiOsit4Δ mutant or the kti12Δ mutant were also found in Elp1 from the wild-type strain  sit4Δ mutant. However, none of the mutations tested altered the zymocin-resistance phenotype of the sit4Δ strain , we also monitored Elongator function via efficiency of ochre suppression mediated by SUP4, a tRNATyr(UUA) that requires Elongator-dependent wobble uridine modification for efficient ochre (UAA) codon readthrough SUP4 and a ura3 ochre allele To investigate the Elp1 C-terminal phosphorylated region, additional combinations of alanine, aspartate or glutamate substitutions at the phosphorylation sites were generated. In addition to using the eclipse assay, we monitored tolerance to intracellular expression of the zymocin tRNase (γ) subunit from the galactose-inducible SUP4-dependent ocura3 suppression, in each case conferring a phenotype comparable to that observed upon complete loss of Elongator function (elp1Δ). In contrast, an S1209D substitution showed considerable restoration of function, while the equivalent glutamate substitution  was less effective. The regain of functionality caused by the phosphomimic aspartate substitution therefore provides evidence that phosphorylation of Ser-1209 acts positively for Elongator function. Although the double S1198A S1202A substitution mutant conferred strong zymocin resistance it showed considerable residual Elongator function in the SUP4 assay, allowing for growth in the absence of uracil similar to that shown by the ELP1 wild-type control. However, concurrent glutamate substitutions at both positions largely restored zymocin sensitivity, supporting the notion that phosphorylation of these two sites acts positively for Elongator function. S1198A S1202A in combination with T1204A S1205A T1206A, a triple alanine substitution that on its own had essentially no effect on Elongator function in any of the assays, conferred stronger zymocin resistance than S1198A S1202A alone and dramatically reduced SUP4-dependent suppression efficiency, indicative of an additive Elongator defect in the quintuple mutant. The triple T1204A S1205A T1206A mutation was used here because phosphorylation at Ser-1205/Thr-1206 could not be unambiguously distinguished (see above). Mutants where acidic residues substituted combinations of these five positions, either alone or in combination with alanine substitutions, indicated that Elongator functionality was not greatly affected by acidic substitutions, although the T1204E S1205D T1206E triple substitution improved Elongator function when combined with the S1198A S1202A double mutant (seen most clearly by the eclipse assay). While the difference in the severity of phenotype observed between the zymocin- and SUP4-based assays for some elp1 mutants might reflect differential effects on tRNAGlu(UUC) and tRNATyr(UUA), we consider it more likely to reflect different loss of modification thresholds required to score positive in these assays; while as little as ∼40% reduction in modification may generate sufficient uncleavable tRNAGlu(UUC) to confer zymocin resistance Tyr(UUA) to support effective ochre suppression. Our results therefore indicate that blocking phosphorylation at all four sites identified in this region of Elp1 leads to reduced Elongator function, with the S1209A mutant showing the greatest defect followed by S1198 S1202A and T1204A S1205A T1206A in decreasing order of severity, but with additivity between S1198 S1202A and T1204A S1205A T1206A leading to a defect as severe as that of S1209A. That acidic substitutions mimicking phosphorylation at each of the four sites conferred considerable Elongator function is consistent with the notion that phosphorylation at these sites functions positively for Elongator.elp1 mutant strains and used LC-MS analysis to quantitate the levels of modified U34 nucleosides. elp1Δ control strain, mcm5U and ncm5U were absent from tRNA as expected. The S1209A and quintuple S1198 S1202A T1204A S1205A T1206A strains showed almost no mcm5U or ncm5U, consistent with the major defect in Elongator-dependent wobble uridine modification indicated by the phenotypic assays  family of protein kinases hrr25 mutants show clear defects in Elongator function in vivo and that they were identified in our in vitro screen due to shared substrate specificity with the related Hrr25 CKI isozyme. Preliminary analysis also failed to generate data supporting a role for Hal5 in Elongator function (We next wished to identify the protein kinase(s) that mediate modification of the phosphorylation sites in the Elp1 carboxy-terminal domain that are important for Elongator function. To take an unbiased approach, we screened a library of 119 GST-protein kinase fusions function ..HRR25 allele (hrr25-I82G) in which the mutant Hrr25 kinase has acquired the capacity to be inhibited specifically by addition of the ATP analogs 1NM-PP1 or 3MB-PP1 hrr25-I82G strain, the Elp1 phosphorylation observed upon incubation of the isolated complex with [γ-32P]ATP was blocked by addition of either 1NM-PP1 or 3MB-PP1  plays a positive role in Elongator function. Previously, we showed that a function ., consisin vivo phosphorylation sites that we have mapped and shown to be relevant for Elongator function: Ser-1198 and Ser-1202. Thus purified Elongator shows Hrr25-dependent phosphorylation that requires the presence of these two residues, while peptides derived from the Elp1 C-terminal region show direct, interdependent phosphorylation by recombinant Hrr25 on Ser-1198 and Ser-1202. Conversely, we found no evidence for direct phosphorylation of Ser-1209 by Hrr25 using three different synthetic peptides containing Ser-1209, or using purified Elongator complex in which Ser-1198 and Ser-1202 were mutated but Ser-1209 was intact.Despite conducting an unbiased, ‘kinome-wide’ screen to identify kinases responsible for Elp1 C-terminal domain phosphorylation, Hrr25, the yeast CKI that associates with Elongator and has already been implicated in Elongator function 1-3 [ST] and [DE]2-4 [X]0-2 [ST], where pS indicates an upstream phosphoserine residue that is needed to prime phosphorylation at the downstream Ser or Thr (shown in bold and underlined) but which can be substituted by an acidic patch in the second class of motif in vitro peptide kinase assays and instead we observed interdependent phosphorylation at these two residues. Furthermore, replacement of one of the two upstream aspartate residues with alanine did not obviously interfere with phosphorylation of Ser-1198 and Ser-1202. Thus although the sites are direct targets of Hrr25 and match the accepted consensus for CKI phosphorylation, dependency of Ser-1202 phosphorylation on prior Ser-1198 modification remains to be demonstrated. Similarly, Ser-1205 and Ser-1209 both fit the pS [X]1-3 [ST] consensus and might be modified once Ser-1202 phosphorylation has occurred, and yet we could not demonstrate Hrr25-dependent incorporation of phosphate at these sites even using a peptide where the upstream sites were already phosphorylated. Given the known substrate specificity of CKIs, it is therefore surprising that we can find no evidence for such priming of phosphorylation at Ser-1205 and Ser-1209 following modification of Ser1202. Furthermore, since the repeating pattern of phosphorylatable residues shown in 1-3 [ST] motif we are even more surprised that we failed to detect phosphorylation of these sites either in vivo or in vitro.Two types of consensus phosphorylation site have been proposed for CKIs: pS [X]hrr25 mutant that is defective for Elongator function strongly supports the involvement of a different kinase at Ser-1209.In spite of the strong evidence we have provided that Hrr25 is a direct Elp1 kinase, inability to detect direct phosphorylation of either the key residue Ser-1209 or of Ser-1205/Thr-1206 by Hrr25 implies that at least one additional Elp1 kinase is involved in Elp1 phosphorylation. While these sites might become better substrates for Hrr25 in the context of fully assembled Elongator complex rather than within synthetic peptides, the fact that we detect normal levels of Ser-1209 phosphorylation in a Although all phosphorylation site mutants examined showed essentially normal assembly of the Elongator complex and retained the ability to interact with the accessory protein Kti12, the S1209A mutation enhanced association of Kti12 with Elongator in comparison with cells expressing either the wild-type protein or the S1198A S1202A mutant. Kti12 stoichiometry is important – either too much or too little interferes with Elongator function hrr25 mutants that cause Elp1 phosphorylation defects and zymocin resistance ATP (3000 Ci/mmol), in the absence or presence of purified recombinant His6-Hrr25 (1 µg). Phosphorylation reactions were stopped by the addition of NuPAGE 4× LDS Sample Buffer (Life Technologies) and heated for 5 min at 95°C. Samples were run on a NuPAGE (4-12%) polyacrylamide/Bis-Tris SDS gel (Life Technologies) at 200 V for 1 h. Gels were stained with Bio-Safe Coomassie (BioRad), dried and subjected to autoradiography. To examine the effect of chemical inactivation of yeast Hrr25 on Elp1 phosphorylation in purified Elongator complex, Elongator was purified as above from strains WAY-H-P1T (wild-type HRR25) and WAY-Has-P1T  and assayed as described above in the presence or absence of 10 µM 3-MB-PP1 or 1-NM-PP1.6-Hrr25, 1 mM ATP and 10 µCi [γ-32P]ATP (6000 Ci/mmol) and incubated at 30°C. Samples (5 µl) were collected at time intervals, spotted on Whatman Grade P81 Ion Exchange Cellulose Chromatography Paper, washed 3 times in 1% phosphoric acid, dried and quantitated by liquid scintillation counting Time course phosphorylation reactions of synthetic peptides were carried out in 150 µl P-buffer containing 500 pmol synthetic peptide (Biomatik), 400 pmol recombinant HisS1 Figin vivo phosphorylation sites in yeast Elp1. Representative MS/MS spectra are shown in which diagnostic B (green) and Y (blue) ions are mapped onto the corresponding peptide sequence. S(Ph), phosphoserine; M(Ox), oxidized methionine. (A) Phosphorylation on Ser-529 within the Elp1 peptide 521-531, parent MH2+ ion m/z 675.29. (B) Dual phosphorylation on Ser-539 and Ser-551 within the Elp1 peptide 532-552, parent MH3+ ion m/z 817.41. (C) Phosphorylation on Ser-636 within the Elp1 peptide 624-652, parent MH3+ ion m/z 1063.46. (D) Phosphorylation on Ser-828 within the Elp1 peptide 828-844, parent MH2+ ion m/z 1051.44. (E) Phosphorylation on Ser-1198 within the Elp1 peptide 1180-1213, parent MH3+ ion m/z 1314.56. (F) Phosphorylation on Ser-1202 within the Elp1 peptide 1180-1213, parent MH2+ ion m/z 1314.56. This spectrum represents a mixture of monophosphorylated isoforms of the peptide: * denotes peaks consistent with Ser-1198 phosphorylation, # indicates peaks consistent with Ser-1205 phosphorylation and ▪ indicates an ion consistent with Thr-1206 phosphorylation. (G) Phosphorylation on Ser-1205 or Thr-1206 within the Elp1 peptide 1180-1213, parent MH2+ ion m/z 1314.56.Mapping (TIF)Click here for additional data file.S2 Fig32P from [γ-32P]ATP in response to GST-kinase fusions identified from the GST-kinase library of Zhu et al. 32P in the absence of added kinase is due to co-purification of Hrr25 with affinity-purified Elongator complex as shown in the main paper. Elp1 runs as a doublet, the faster-migrating component of which is truncated at the amino-terminus http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the yeast proteome with Hal5 using the default parameters indicates that Kkq8 and Sat4 are its closest relatives within the yeast kinome. (C) Deletion of HAL5, either alone or in combination with KKQ8 and/or SAT4, fails to confer zymocin sensitivity by eclipse assay.Identification of Elp1 kinases. (A) Phosphorylation of yeast Elongator by yeast protein kinases. Elongator was purified from yeast using TAP-tagged Elp3 and incorporation of (TIF)Click here for additional data file.S3 Fighrr25-3 mutant strains. Elp1 phosphorylation in samples of extract containing equivalent amounts of protein was monitored by Western blot analysis using the phosphoSer-1209 phosphospecific antibody.Elp1 shows similar levels of Ser-1209 phosphorylation in wild-type and (TIF)Click here for additional data file.S4 Figin vitro phosphorylation of a peptide corresponding to Elp1 residues 1193–1213. MS/MS spectra are shown in which diagnostic B (green) and Y (blue) ions are mapped onto the corresponding peptide sequence. S(Ph), phosphoserine. (A) Phosphorylation on Ser-1198, parent MH3+ ion m/z 889.08 corresponding to monophosphorylated peptide. * denotes peaks consistent with phosphorylation on Ser-1202, suggesting that this is a mixed spectrum. (B) Phosphorylation on Ser-1202, parent MH3+ ion m/z 889.08 corresponding to monophosphorylated peptide. * indicates peaks consistent with phosphorylation of Ser-1198, suggesting that this is a mixed spectrum. (C) Phosphorylation on Ser-1198 and Ser-1202, parent MH3+ ion m/z 925.73 corresponding to diphosphorylated peptide. * denotes peaks consistent with one phosphate on Ser-1202 and a second phosphate to the right, most likely on Ser-1205.Mapping of phosphorylation sites following (TIF)Click here for additional data file.S5 Figelp1Δ), YCplac111-ELP1-6HA (ELP1 wild-type) or its Thr-1212 mutant derivatives as indicated. Zymocin sensitivity was measured by eclipse assay Click here for additional data file.S6 FigAlignment of Hrr25 phosphorylation sites. Alignment of the Elp1 Ser-1198 phosphorylation site with mapped Hrr25 phosphorylation sites in Tif6 (TIF)Click here for additional data file.S7 Fighrr25-2 leads to increased association of Hrr25 and Kti12 with Elongator. Elp1-HA was immunoprecipitated from extracts of the indicated HRR25 and hrr25-2 strains and immunoprecipitates were examined by Western blotting with anti-HA, anti-myc and anti-Hrr25 antibodies to detect immunoprecipitated Elp1 and co-immunoprecipitated Kti12 and Hrr25 respectively. The ELP1 untagged control confirms that recovery of Kti12 and Hrr25 is dependent on Elp1.(TIF)Click here for additional data file.S1 Tableelp1 alleles. WAY034 (elp1Δ) and WAY031 (elp1Δ sit4Δ) were transformed with YCplac111, YCplac111-ELP1-6HA or with mutant derivatives of the latter plasmid and monitored by Eclipse assay for Zymocin sensitivity/resistance. R, Zymocin resistant; S, Zymocin sensitive; R/S, intermediate.Summary of Zymocin phenotype monitored by Eclipse assay associated with different mutant (PDF)Click here for additional data file.S2 TablePlasmids used or generated in this study.(PDF)Click here for additional data file.S3 TableYeast strains used or generated in this study.(PDF)Click here for additional data file.


Tgr5 knockout mice. Together, these results demonstrate that compound 18 is a mouse-selective TGR5 agonist that induces GLP-1 and PYY secretion, and lowers the glucose excursion in an OGTT, but only at doses that simultaneously induce gallbladder filling. Overall, these data highlight the benefits and potential risks of using TGR5 agonists to treat diabetes and metabolic diseases.Activation of TGR5 via bile acids or bile acid analogs leads to the release of glucagon-like peptide-1 (GLP-1) from intestine, increases energy expenditure in brown adipose tissue, and increases gallbladder filling with bile. Here, we present compound 18, a non-bile acid agonist of TGR5 that demonstrates robust GLP-1 secretion in a mouse enteroendocrine cell line yet weak GLP-1 secretion in a human enteroendocrine cell line. Acute administration of compound 18 to mice increased GLP-1 and peptide YY (PYY) secretion, leading to a lowering of the glucose excursion in an oral glucose tolerance test (OGTT), while chronic administration led to weight loss. In addition, compound 18 showed a dose-dependent increase in gallbladder filling. Lastly, compound 18 failed to show similar pharmacological effects on GLP-1, PYY, and gallbladder filling in  In 1902, Bayliss and Starling speculated that an endocrine substance arising from the gut after ingestion of nutrients induces secretions from the pancreas . In factq-coupled receptors  for fatty acids and Gs-coupled receptors (e.g. GPR119) for fatty acid derivatives  compared to the high exposure of INT-777 dosed at 100 mg/kg   , but a nvehicle) . Conversvehicle) , as wellvehicle) . Lastly,3 μM·hr] . TogetheDue to the strong GLP-1 and PYY stimulation of compound 18 in C57BL/6 mice, we hypothesized that compound 18 would improve glucose tolerance. To test this, we performed OGTTs in C57BL/6 mice treated orally with either a dose-range of compound 18, a single dose (100 mg/kg) of INT-777, or a single dose (10 mg/kg) of the DPP4 inhibitor sitagliptin  as a positive control. An oral challenge of glucose (3 g/kg) was given 15 min after compound administration since compound 18-induced peaks in GLP-1 were observed at this time-point. INT-777 had no detectable improvement of glucose tolerance despite substantially higher plasma exposure , while cWhile compound 18 showed robust GLP-1 and PYY stimulation in fasted mice , and lowWhile compound 18 led to significant glucose lowering in an OGTT studies when dosed acutely, it remained unclear if the mechanism of TGR5-stimulated GLP-1 secretion desensitizes over time. In addition, oral dosing of both bile acid (cholic acid) and INT-777 has been previously reported to result in weight loss in diet-induced obese (DIO) mice with chronic administration ,22. In owild-type but not Tgr5 KO mice, suggestive of a TGR5-dependent mechanism , it was still sufficient to cause gallbladder filling at this dose / exposure. A true gut-restricted and efficacious TGR5 agonist has yet to be described. Overall, these results using a highly selective and potent small molecule agonist of TGR5 demonstrate the magnitude of TGR5 agonists to stimulate GLP-1 and PYY secretion in vivo, the likelihood of TGR5 agonists to lead to weight loss in chronic studies, and the potential of TGR5 agonists to lower glucose. Unfortunately, these results also demonstrate the physiological consequences of TGR5 agonism at the gallbladder, confirming the studies performed by Li et al. [One potential way to avoid TGR5 agonist–induced gallbladder filling is the development of gut-restricted TGR5 agonists, allowing stimulation of the intestinal L-cells to produce GLP-1 and PYY, while reducing or eliminating exposure of an agonist to the gallbladder. Unfortunately, while compound 18 had low systemic exposure [Ci et al. . Importa


P < 0.05) on 16S rRNA gene diversity revealed significant differences (P < 0.05) between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage FI did not differ significantly from the RW and the M vegetated sites. These results pointed out the effect of different floristic communities rhizospheres on their soil bacterial communities.The rhizosphere effect on bacterial communities associated with three floristic communities  which differed for the developmental stages was studied in a high-altitude alpine ecosystem. RW site was an early developmental stage, FI was an intermediate stage, M was a later more matured stage. The N and C contents in the soils confirmed a different developmental stage with a kind of gradient from the unvegetated bare soil (BS) site through RW, FI up to M site. The floristic communities were composed of 21 pioneer plants belonging to 14 species. Automated ribosomal intergenic spacer analysis showed different bacterial genetic structures per each floristic consortium which differed also from the BS site. When plants of the same species occurred within the same site, almost all their bacterial communities clustered together exhibiting a plant species effect. Unifrac significance value ( A glacier foreland after glacier retreat can be considered a cold desert, being composed of habitats characterized by severe climatic regimes and barren substrate with low total carbon and nitrogen contents . Rock cr Poa alpina L. was strongly influenced by the environmental conditions but, in transition and mature stage, plants could select a specific microbial community [ Leucanthemopsis alpina (L.) Heywood showed an opposing rhizosphere effect with a specific microbial community in the early successional stage only [ Lc. alpina on the microbial community and on the physical-chemical parameters in an early successional stage  did not exhibit significant trends, supporting the conclusion of Tscherko et al. [Furthermore, pioneer plants could select rhizosphere microbial communities able to promote plant growth thanks to the interactions in nutrient cycling and carbon sequestration . Neverthommunity . Along aage only . The stuo et al. .However, in a safe-site, the pioneer vegetation interrelated in floristic consortia often exhibited ground stems and root tangle with large nets. In this case, a safe-site could be equaled to a transitional or even a mature grassland for root tangle and plant community structure. The floristic community effect in such a habitat was observed in natural as well as in artificial experimental sites . Osanai  Stipa, Hilaria, and for the invading annual grass Bromus [On the other hand, studies about the effect of plant cover on microbial community in cold environments regarded different ecological niches and pointed out the higher significance of environmental parameters than the influence of the floristic consortia. In Antarctic environments along a latitudinal gradient, bacterial diversity of dense vegetation from different locations was comparable whereas bacterial diversity of “fell-field” vegetation decreased with increasing latitude , 18. In s Bromus .Consequently, the aim of this work was to investigate if, in different safe-sites on a deglaciated terrain of the same chronosequential age, floristic consortia could select specific rhizobacterial communities.2. The main geological processes are periglacial and the streamflow is characterized by the glacier dynamics. During 1970–2000, the valley had an average rainfall of about 550 mm per year. In 2011, the mean temperatures of the plant growing season were 7.3°C in July, 10.3°C in August, and 8°C in September and the mean precipitations were 2.7, 2.5, and 3.6 mm per day, respectively. The dominant rock types are schist and gneiss [2 left after a quick glacier retreat in the last 160 years [The study site is located in the upstream subcatchment of Saldur river  in the high Matsch valley  with a drainage area of 11 kmd gneiss  and the 60 years , was loc Diphasiastrum alpinum and Gnaphalium supinum L.; FI site, a floristic island between big rocks, was colonized by Cladonia sp., Festuca halleri All., Polytrichum sp., Racomitrium sp., Sedum alpestre Vill., and Senecio carniolicus (Willd.) Braun-Blanq.; M site, a safe-site surrounded by big rocks and characterized by a flatter area, was colonized by Cetraria islandica (L.) Ach., Leucanthemopsis alpina (L.) Heywood, Potentilla aurea L., Rhododendron ferrugineum, Sibbaldia procumbens L., and Silene acaulis (L.) Jacq. These sampling sites were carefully chosen in order to share similar conditions in terms of altitude, features, and geology.Rhizosphere and soil sampling were carried out in 2011 May, at the beginning of the plant growing season. Three safe-sites  characterized by loosely organized assemblages of different plant species and a bare soil (BS site) were sampled. The sites were less than 20 × 20 cm. RW site, below an iron rich rock-face, was colonized byThe rhizosphere samples of all the single plant individuals within a floristic community were collected. Each individual plant was carefully pulled out the soil, without damaging its single root system. After pulling out each plant and avoiding roots, 4 g of rhizosphere soil strictly adhering to the roots was collected with a pair of sterile tweezers. Three replicates of bulk soil were collected as a control. Moreover, from each safe-site, 50 g of root-free soil was collected and put into plastic bags for soil chemical analysis. All the samples were immediately transported in refrigerated boxes to the laboratory as soon as the logistic constraints permitted and they were stored at −80°C until analysis.3 concentrated 65% and H2O2 30%) in a milestone high performance microwave oven . To determine the total organic carbon content, soil samples were also acidified with HCl (6 M) to eliminate all carbonates. Metals and total phosphorous were determined by inductively coupled plasma-optical emission spectroscopy . Nitrogen and C were quantified with an elemental analyzer . The pH2OH was measured using an Accumet AP85 pH . To test the level of significance of the observed chemical differences among sites, a Kruskal-Wallis test was done by using Mann-Whitney pairwise comparison post hoc test and Bonferroni correction in Past software [Soil samples for chemical analysis were oven-dried at 105°C until constant weight and then acid was digested . Microbial analyses were carried out using denaturing gradient gel electrophoresis (DGGE)  to descrFor DGGE analysis, primers GC357f and 907r were used as described . DGGE waARISA fingerprint was performed as described by Cardinale et al.  with thex/∑x)∗1000, where “x” is the fragment height in units of fluorescence, and then transformed on a logarithmic scale for multivariate analysis. Log-transformation was used to stabilize the sample variance, to reduce the interaction effect, and to normalize the distribution of data. Moreover, log-transformation can combine the information of a binary matrix with those of a nontransformed data matrix, hence preserving the relative abundance information and down-weighting dominant groups.ARISA matrix was normalized with the formula  was applied with Bray-Curtis algorithm. NMDS does not need the assumption of linear associations among variables being described as the most efficient ordination method for microbial ecology . Bray-CuThe Nexus format of the phylogenetic tree of the DGGE identified bands performed by MEGA5 was submitted to the UniFrac web server to test differences among sites based in the UniFrac metric with 100 permutations and the Bonferroni correction factor . A princP < 0.05; data not shown).Soil resulted to be a sandy silt soil with an average texture of 72.3 ± 5.0% of sand, 21.0 ± 4.1% of silt, 6.6 ± 1.3% of clay, and 4.6 ± 1.3% of humus; pH was 4.5 ± 0.3%. Average chemical composition of sampled soils was total P 0.7 ± 0.1 mg/kg d.m., total K 7.4 ± 1.0 mg/kg d.m., total Ca 3.4 ± 0.6 mg/kg d.m., total Mg 13.4 ± 1.7 mg/kg d.m., total Fe 45.4 ± 6.9 mg/kg d.m., and total Al 29.4 ± 5.6 mg/kg d.m. No calcium carbonate was detected. Since those safe-sites were located in proximity of each other, their soil chemical composition did not differ substantially between sites  to the most vegetated M site (0.98% dry weight) and also total organic carbon grew up from BS site (0.62% dry weight) to M site  and the performed test showed significant differences in the pairwise comparisons of the sites with R values approaching 1 in most of the cases . In FI and M sites the tendency of individuals of the same species to cluster together seems to disappear, except for R. ferrugineum, maybe due to the higher number of species interconnected in the safe-site.Due to the high sensitivity of the automated capillary electrophoresis, ARISA fingerprints of both rhizosphere and bare soil bacterial communities provided complex profiles with peaks ranging from 151 bp to 1437 bp and the 16S-23S rRNA internal transcribed spacer region (ITS) richness varied from 43 to 168 peaks. The electropherograms, characterized by distinct peaks number and intensity, revealed a shift in bacterial community structure across the different safe-sites plant communities. On the NMDS plot , samples from root-free soil (BS), safe-site of early developmental stage (RW), intermediate stage (FI), and from the most mature one (M) showed four separate clusters based on microbial community structure . Accordihe cases . Where r Gp3 and Gp1, Sphingobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinobacteria  showed that the BS site samples were significantly different from FI and M sites rhizospheres, but not from RW site rhizospheres. Moreover, the FI site rhizosphere did not differ significantly from M and RW sites rhizospheres, while the M site rhizospheres exhibited significant differences with the RW site. A PCoA analysis of the UniFrac distance matrix was calculated to assess the overall sequence population similarity among safe-sites  were found. We discovered a distinct clustering of bacterial communities according to RW, FI, and M vegetation types that are significantly diverse from the unvegetated soil (BS site). We also found that a gradient in terms of C and N enrichments from BS site to the most developed M site was an important determinant of microbial community profiles. UniFrac analysis showed site-shifts in bacterial diversity which suggest a specialized physiology adapted to the peculiar site environmental conditions. Moreover, the differences among safe-sites, according to C and N gradients, support the occurrence of a plant cover effect on the rhizosphere bacterial community within those safe-sites.The vegetation complexity of the three safe-sites  raised from a simple colonization of two species (RW site) to the colonization of lichens, mosses, and few herbaceous plant species (FI site) till M site, where five herbaceous species and one woody species  of a glacier forefield showed no selective effect on the microbial community, since a similar bacterial community structure was apparent up to 40 cm of distance to the plant [ P. alpina, did not exhibit a selective role on its rhizosphere bacterial community in the pioneer stage of a chronosequence, maybe due to the harsh environmental conditions of the plot where it was growing [Previous investigations of the rhizosphere effect were conducted on few single pioneer plants or in grassland plots. Almost all the researches on the rhizosphere effect associated with a single plant species were achieved on crop or other plants either in artificial microcosms such as pots or on agricultural soils such as orchards and crop monocultures. Most of these researches demonstrated that peculiar root exudation and rhizodeposition of different plant species could select the structural and functional diversity of the associated rhizosphere bacterial communities –36. On ttructure . In anothe plant . Another growing . HoweverOn the other hand, safe-sites are more complex than single pioneer plant individuals in a cold environment, but they show less complexity than a homogeneous plot carefully designed in mountain grasslands. Real safe-sites are much less homogeneous, being shaped by the history of the microarea where they are such as dynamical differences in climate, in geophysical features, or in biota colonization which determine complicated patterns and often unique rates of soil development . In our The rhizosphere bacterial communities of RW site, characterized by only two different plant species, clustered more closely with the BS site than with the vegetated ones showing a simpler bacterial community, as confirmed by the UniFrac analysis which detected no significant difference between the two sites. Although FI and M sites had a similarity of about 56%, inside the FI site were found rhizobacterial communities of mosses and lichens which did not cluster strictly with the plant ones. The presence of lichens and mosses in the same site could explain why the bacterial community of the FI site represented an intermediate stage between the RW site and the M site. The M site, colonized by individuals of six plant species, could be considered a later stage where floristic consortia selected a more complex bacterial community which significantly differs from the one of BS and RW sites. The UniFrac analysis showed that the BS communities were distinct from ones of the FI and M sites and were weakly similar to the ones of RW site. Moreover, the intermediate plant colonization stage, FI site, did not differ significantly from the RW and the M vegetated sites. Previous studies , 45, 46 Despite the harsh environmental condition of the natural alpine ecosystem and the tight complex root system of the safe-site, our results support the capability of different pioneer plant consortia to select specific rhizobacterial communities with an increase of bacterial diversity according to the increase of soil maturation. Moreover, when plants of the same species occurred in the same site, the associated rhizobacterial communities clustered more strictly together according to their genetic structures, confirming the high similarity of the rhizobacterial communities within individuals of the same pioneer plant species.


Progressive loss of skeletal muscle, termed muscle wasting, is a hallmark of cancer cachexia and contributes to weakness, reduced quality of life, as well as poor response to therapy. Previous studies have indicated that systemic host inflammatory response regarding tumor development results in muscle wasting. However, how tumor directly regulates muscle wasting via tumor-derived secreted proteins is still largely unknown.IGFBP3, which encodes IGF-binding protein-3 (IGFBP-3), is dramatically up-regulated in pancreatic tumor samples. We also verified the wasting effect of IGFBP-3 on C2C12 muscle cells with biochemical and genetic assays.In this study, we performed bioinformatics analysis in two datasets of pancreatic ductal adenocarcinoma, which causes cancer cachexia and muscle wasting with the highest prevalence, and uncovered that IGFBP3 knockdown in Capan-1 cells or IGFBP-3 antibody neutralization. Strikingly, compared to muscle cells, IGF signaling and proliferation rate of Capan-1 cells were rarely affected by IGFBP-3 treatment.IGFBP-3 potently leads to impaired myogenesis and enhanced muscle protein degradation, the major features of muscle wasting, via IGF signaling inhibition. Moreover, conditioned medium from Capan-1 pancreatic cancer cells, which contains abundant IGFBP-3, significantly induces muscle cell wasting. This wasting effect is potently alleviated by Our results demonstrated that pancreatic cancer cells induce muscle wasting via IGFBP-3 production.The online version of this article (doi:10.1186/s13046-016-0317-z) contains supplementary material, which is available to authorized users. Patients with pancreatic cancer often develop the most severe degrees of cachexia that is highly associated with cancer death . ClinicaDrosophila  tyrosine (PerkinElmer) incorporation into C2C12 myotube cultures. After treatment of indicated reagents for 24 h, the C2C12 myotubes were incubated with differentiation medium containing 5 μCi/ml [3H] tyrosine for 2 h. The medium then was discarded, cells were washed twice with PBS, and 1 mL of 10 % trichloroacetate was added to each well. Total cell lysates were centrifuged and the pellets were washed with 95 % ethanol and dissolved in 0.1 M NaOH. Samples were analyzed for total radioactivity and protein concentration using a scintillation counter (PerkinElmer) and the Bradford assay, respectively. The final radioactivity was normalized to protein level. As for protein degradation, C2C12 myotubes were incubated with 5 μCi/ml [3H] tyrosine for 48 h to label cellular proteins. Afterward, myotubes were incubated with medium containing 2 mM unlabeled tyrosine and indicated reagents control for 24 h. The medium was collected, precipitated with 10 % trichloroacetate and centrifuged. The acid-soluble radioactivity, which reflects degraded protein level, was measured using a scintillation counter.Total protein synthesis was assessed quantifying the amount of [t test in Microsoft Excel. The values were presented as means ± SEM and significance was defined as * P < 0.05.Statistical analysis was performed using Student’s


Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling.Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike  Current methods to measure post-translational modifications of receptor tyrosine kinases provide ensemble results. Here the authors develop a single-molecule blotting (SiMBlot) assay that detects site-specific phosphorylation patterns at the single-molecule level. Receptor tyrosine kinases (RTKs) play important roles in diverse biological functions, including cell-to-cell communication, proliferation and signal propagation, by their inherent kinase activity. Most biological processes, including RTK signaling, are coordinated by protein regulation such as post-translational modifications (PTMs), many of which provide binding sites for specific protein–protein interactions and signaling complex formation13489Recently, the emerging development of single-molecule techniques enables the observation and characterization of individual molecules for exquisite qualitative and quantitative analysis, avoiding ensemble error111213131511Single-Molecule Blotting' (SiMBlot). SiMBlot can directly immobilize biotinylated cell surface proteins on the single-molecule surface and enables the pairwise immunofluorescence labeling to detect multi-site PTMs of a single polypeptide molecule. To demonstrate the unique power of this approach, we apply SiMBlot to reveal the pairwise site-specific phosphorylation patterns of individual EGFR molecules, which are extracted from the cell surface membrane in response to the EGF stimulus or sampled from an in vitro autophosphorylation assay. Our results call into question ligand-dependent multi-phosphorylation of EGFR, which is popularly believed to occur12Here, we have described a simple, ultra-rapid and low-cost single-molecule assay with an antibody-free immobilization to investigate combinatorial PTMs of RTKs, named as ‘In previously reported single-molecule isolation techniques1119Since cell surface biotinylation may affect the physiological function of PM-loaded receptor proteins, we examined the biological function of biotinylated membrane proteins by using conventional western blot analysis for RTK signaling. We observed identical EGF-dependent tyrosine phosphorylations of unlabeled and biotinylated EGFR  as an in−1) by SiMBlot and immunoblotting, as an ensemble analysis. As reported previouslyMoreover, to test whether SiMBlot can reflect the results of a conventional method for site-specific modifications, we measured site-specific phosphorylation of EGFR in response to EGF ref. To confirm the ability of SiMBlot to directly visualize pairwise site-specific modifications of individual proteins, we used EGF-dependent phosphorylated EGFR. Since EGF-induced multiple phosphorylation of EGFR has been suggested by enormous ensemble biochemical results and it has long been widely believed12in vitro rEGFR kinase assay to phosphorylate most tyrosine residues in its C-terminal tail, as evidenced by the detection of autophosphorylation by immunoblotting (in vitro autophosphorylated rEGFR with pairwise autophosphorylation antibody sets (pTyr845/pTyr1173 and pTyr1068/pTyr1173). After immobilization of rEGFR ; Lift values  are approximately equal to 1  of the polypeptide chain to be examined. However, SiMBlot can be easily applied to analyze even the endogenously modified proteins taken from cells by probing for PTM sites dispersed along the entire protein length using available site-specific primary antibody and fluorescently labeled secondary antibody.Besides MS-based proteomics, recently, the nanopore and nanogap methods in nanotechnology are rapidly developing but remain incompatible for single-molecule proteomic analysis of PTMs13Although we only report the application of this method to combinatorial PTMs of EGFR, previous studies20331Antibodies against EGFR, phospho-EGFR , and phospho-Erk were purchased from Cell Signaling Technology . A monoclonal anti-pTyr845 EGFR antibody was purchased from Millipore (Cat. #04-283). A monoclonal anti-β-actin antibody was purchased from MP Biomedicals (Cat. #08691001). An Alexa488-labeled antibody and Lipofectamine were purchased from Invitrogen Life Technology . A Cy3-labeled secondary antibody was purchased from Jackson ImmunoResearch Inc. (Cat. #111-165-003). Recombinant human EGF was purchased from R&D Systems (Cat. #236-EG). Sulfo-NHS-Biotin was purchased from Thermo Scientific (Cat. #21217). Anti-FLAG M2 affinity gel, 3 × FLAG peptides, and Phosphatase Inhibitor Cocktail 3 were purchased from Sigma-Aldrich (Cat. #A2220 and #F4799). Phosphopeptides  were purchased from PEPTRON Inc.2-controlled (5%) incubator. CHO-K1 cells were maintained in Ham's F-12K medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified CO2-controlled (5%) incubator. For transfection and transient expression of proteins, cells were transfected with plasmids encoding rEGFR using Lipofectamine . Transfections were performed using Lipofectamine (according to the manufacturer's instructions) and cells were then cultured for an additional 48 h to achieve ectopic expression of rEGFR.COS7 and A431 cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified COTCTAGA GGAGGG ATGGTGAGCAAGGGCGAGGAG -3′, reverse: 5′- C ACCGGT TCA CTTGTCGTCATCGTCTTTGTAGTC CTTGTACAGCTCGTC -3′). The constructs for WT and mutant rEGFR  were cloned by inserting the N-terminus of the corresponding cDNA sequence of whole EGFR into pcDNA3.1 eGFP-flag using EcoRI/XbaI without its own stop codon.A plasmid encoding C-terminal flag-tagged eGFP was constructed by insertion of the corresponding cDNA sequences of eGFP into the XbaI/AgeI sites of the pcDNA 3.1 myc/his plasmid (Invitrogen). The inserted DNA fragment encoding C-terminal flag-tagged eGFP was prepared by PCR using the pEGFP-C1 vector (Clontech Laboratory Inc.) as the template and primers  prepared in PBS was added. Cells were incubated with gentle agitation for 20 min at 4 °C and further incubated with 0.1 M glycine prepared in PBS for 10 min at 4 °C. After washing with serum-free Dulbecco's Modified Eagle's Medium, cells were treated with a ligand such as EGF at 37 °C for 10 min, the medium was removed, and cells were washed with ice-cold PBS. Cells were lysed in lysis buffer  by sonication and cell lysates were centrifuged at 13,500 rpm, 4 °C for 10 min. Supernatants were prepared for single-molecule imaging or subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblotted.Cell surface labeling was conducted as previously described, with minor modifications2, 5 mM β-glycerophosphate, 0.5 mM dithiothreitol and 0.1 mM sodium orthovanadate). Immunoprecipitates were eluted from beads with 0.1 mg ml−1 3 × FLAG peptide at 4 °C for 30 min. Purified rEGFR was incubated for 1 h at 30 °C with shaking. EGFR was activated by adding 0.1 mM ATP and 100 ng ml−1 EGF prepared in kinase buffer. The reaction was stopped by adding 2% SDS. Samples were boiled for 10 min and subjected to single-molecule imaging or immunoblotting.COS7 cells ectopically expressing rEGFR were serum-starved for 12 h. After cell surface biotinylation and cell lysate preparation using lysis buffer , rEGFR 2O and 1 M KOH for 2 h or longer, and treated with 3-(2-aminoethylamino)-propyltrimethoxysilane  and doped with mPEG and biotin-PEG  with the mass ratio of 25:1 (mPEG:biotin-PEG)−1 NeutrAvidin  ref. Extensively cleaned cover glasses were prepared by cleaning with H2, 0.05% (v/v) Tween-20 and 0.1 mg ml−1 bovine serum albumin, pH 8.0). Unbound antibodies and samples were removed from the channels by washing two times with buffer SB18. For immunofluorescence detection, immobilized protein molecules were incubated with a different antibody (13.3 nM) against the prey protein for 10 min and with a fluorescent dye-labeled secondary antibody (2.66 nM) for 10 min immediately before imaging.For pairwise antibody probing, samples were denatured by boiling for 5 min with 2% SDS. Samples were serially diluted to obtain well-isolated spots on the NeutrAvidin-coated surface upon incubation for 10 min. All dilutions were made immediately before experiments with buffer SB18  and standard deviation were calculated from images of five or more different regions.An objective-type TIRF microscope  was used to acquire single-molecule dataColocalization between Alexa488 and Cy3 was assessed as described previously, with minor modificationst-test.All data are presented as the mean±s.d. or representative images of at least three sets of independent experiments. When necessary, data were statistically analyzed using the Student's How to cite this article: Kim, K. L. et al. Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting. Nat. Commun. 7:11107 doi: 10.1038/ncomms11107 (2016).Supplementary Figures 1-15 and Supplementary Tables 1-3


Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease. Cardiac troponin I (cTnI) and its binding partner cardiac troponin T (cTnT) are the biomarkers of choice for the diagnosis of myocardial damage , 2. Upon2+ [2+]), cTnI binds actin at multiple sites maintaining tropomyosin at the outer domain of actin, and thereby blocks myosin-binding sites on the thin filament and prevents force development. In systole (high intracellular [Ca2+]), Ca2+ binds to cTnC and induces a conformational change in the troponin complex. This results in the release of cTnI from actin and a shift of tropomyosin closer into the grove of the actin filament, thereby enabling actin-myosin interactions and cardiomyocyte force development ..2+ sensiStudying cTnI phosphorylation in different disease phenotypes and at different disease stages will help us to gain a better understanding of the role of PKC-mediated cTnI phosphorylation in cardiac disease. Additionally, with this increasing knowledge about cTnI phosphorylation in cardiac disease, cTnI phosphorylation patterns at the different PKA- and PKC-phosphorylation sites may become promising biomarkers since cTnI phosphorylation status can be determined from blood samples after acute cardiac insult.


Despite their indispensable roles in sensory processing, little is known about inhibitory interneurons in humans. Inhibitory postsynaptic potentials cannot be recorded non-invasively, at least in a pure form, in humans. We herein sought to clarify whether prepulse inhibition (PPI) in the auditory cortex reflected inhibition via interneurons using magnetoencephalography. An abrupt increase in sound pressure by 10 dB in a continuous sound was used to evoke the test response, and PPI was observed by inserting a weak (5 dB increase for 1 ms) prepulse. The time course of the inhibition evaluated by prepulses presented at 10–800 ms before the test stimulus showed at least two temporally distinct inhibitions peaking at approximately 20–60 and 600 ms that presumably reflected IPSPs by fast spiking, parvalbumin-positive cells and somatostatin-positive, Martinotti cells, respectively. In another experiment, we confirmed that the degree of the inhibition depended on the strength of the prepulse, but not on the amplitude of the prepulse-evoked cortical response, indicating that the prepulse-evoked excitatory response and prepulse-evoked inhibition reflected activation in two different pathways. Although many diseases such as schizophrenia may involve deficits in the inhibitory system, we do not have appropriate methods to evaluate them; therefore, the easy and non-invasive method described herein may be clinically useful. Therefore, their actual function in a circuit largely remains unknown. Information on cortical inhibition in humans is very limited. Since many diseases including epilepsy [Cortical neural circuits are composed of excitatory pyramidal cells (PCs) and inhibitory interneurons. In order to understand how circuits process information, their functions as well as the balance between them have to be elucidated. However, the roles of inhibitory interneurons have not been examined in as much detail as PCs. One reason is their great diversity in morphology, connections, intrinsic electrophysiological properties such as firing, synaptic dynamics including their excitatory inputs, subcellular domains targeting, and neuropeptide expression –9; thereepilepsy ,11, schiepilepsy , depressepilepsy , bipolarepilepsy , panic depilepsy , autism epilepsy , and essepilepsy  may haveInhibitory postsynaptic potentials (IPSPs) cannot be recorded non-invasively, at least in a pure form, which is an important problem, particularly in humans. As a candidate for an indirect method to measure inhibitory processes, we recently developed a technique by which to observe prepulse inhibition (PPI) in the human cortex ,19. ConvIn the present study, we sought to clarify whether the PPI of change-related cortical responses represented an active inhibitory process, and also whether the time course of the inhibition was consistent with those of IPSPs. For the former, we examined whether the magnitude of inhibition depended on the magnitude of prepulse-evoked brain responses. If suppression of the test response depends on events in excitatory synaptic transmission exclusively, the degree of inhibition should depend on the magnitude of prepulse-evoked excitatory responses. For the latter, we employed a paired-pulse stimulation paradigm with various conditioning-test intervals from 10 to 800 ms. By manipulating the timing of the prepulse, we can estimate the time course of inhibitory activity of the prepulse. The time course of IPSPs is well known in whole-cell patch clamp studies consisting of an early component peaking at 20–30 ms (e.g. ) and a lThis study was approved in advance by the Ethics Committee of the National Institute for Physiological Sciences, Okazaki, Japan, and written consent was obtained from all subjects. The experiment was performed on 13  healthy volunteers, aged 24–51 (35.2 ± 8.7) years. They were asked to refrain from alcohol, caffeine, and smoking for at least 12 hours prior to the experiment. There were three smokers. None of the subjects had any history of mental or neurological disorders or substance abuse in the last two years. They were free of medication at testing. They had a hearing threshold lower than 30 dB at 1000 Hz as assessed by an audiometer .Repeats of a 1-ms sine wave click at 100 Hz  were useMagnetic signals were recorded using a 306-channel whole-head type MEG system , which comprised 102 identical triple sensor elements. Each sensor element consisted of two orthogonal planar gradiometers and one magnetometer coupled to a multi-superconducting quantum interference device (SQUID), and thus provided 3 independent measurements of the magnetic fields. In the present study, we analyzed MEG signals recorded from 204 planar-type gradiometers. These planar gradiometers are sufficiently powerful to detect the largest signal just over local cerebral sources. Signals were recorded with a bandpass filter of 0.1–300 Hz and digitized at 1004 Hz. An analysis was conducted from 100 ms before to 300 ms after the onset of the Test (Test-evoked response) or the onset of the Prepulse (Prepulse-evoked). Epochs with MEG signals larger than 2.7 pT / cm were rejected from the averaging.Experiments were conducted in a quiet, magnetically shielded room. Subjects sat in a chair and watched a silent movie on a screen 1.5 m in front of them throughout the experiments. Five experiments: Experiments 1–1 ~ 1–4 and Experiment 2, were carried out on different days. In all experiments except Experiment 1–3, all 13 subjects were tested. Four subjects did not participate in Experiment 1–3 because of their personal circumstances.The effects of the Prepulse-Test interval were examined from 10 to 200 ms. The Standard sound was 600 ms in length. The Prepulse was presented either 10, 30, 60, 100, 150, or 200 ms before the Test presented at 400 ms in the click train. Therefore, there were 14 stimuli: 1) Standard, 2) Test alone, 3)–8) Prepulse alone, and 9)–14) Prepulse + Test. The fourteen stimuli were presented randomly at an even probability at a trial-trial interval of 1000 ms. A total of 120–125 artifact-free epochs were averaged for each stimulus.Recorded MEG waveforms were subjected to band-pass filtering of 1–75 Hz and analyzed as previously reported ,19. In bThe effects of longer Prepulse-Test intervals were explored using a 75-dB click (Prepulse) presented either 300, 400, 500, 600, 700, or 800 ms before the Test at 1000 ms in the click train. The Standard sound was 1200 ms in length. Unlike the shorter intervals in Experiment 1–1, responses to the Test and Prepulses did not temporally overlap in this recording. In addition, compared to Experiment 1–1, a markedly longer time was needed to complete the recording. Therefore, in Experiments 1–2 and 1–3, there was only one Prepulse-alone condition, in which a prepulse was presented 400 ms before the Test. The Prepulse + Test response were evaluated without subtraction procedures  as descrSince the results of Experiment 1–2 showed significant inhibition for the Prepulse at 600 ms only, we considered it necessary to confirm the inhibition itself and the time course of the inhibition further using stronger prepulses. All the procedures in Experiment 1–3 were the same as those in Experiment 1–2, except that the prepulse used was three consecutive clicks. For example, the Prepulse inserted 600 ms before the Test was composed of three clicks of 75 dB at 600, 590, and 580 ms before the Test . It was Short-latency inhibition was examined in more detail using prepulses presented either 10, 20, 30, 40, 50, 60, or 70 ms before the Test. Other than the prepulse timing, the procedures of recordings and analyses were similar to those in Experiment 1–1.The effects of prepulses with a train of clicks were examined. There were five prepulses: a click of 75 dB presented at 60 ms before the onset of the Test (1-Prepulse), two clicks at 60 and 50 ms (2-Prepulse), three clicks at 60, 50, and 40 ms (3-Prepulse), four clicks at 60, 50, 40, and 30 ms (4-Prepulse), and two clicks at 60 and 30 ms (6030 Prepulse) . The TesThe peak amplitude of Change-N1m was measured between the peak in Change-N1m within the period of 100–200 ms and the peak of the polarity-reversed earlier component within 50–100 ms ,32. The The test stimulus (Test), an abrupt increase in sound pressure by 10 dB in a continuous click train at 100 Hz and 70-dB SPL , evoked 6,72 = 13.06, p = 6.2 x 10−10, partial η2 = 0.52), but Hemisphere was not . In Experiment 1–1, the effects of the Prepulse-Test interval were examined in order to explore the time course of inhibition by inserting the Prepulse 10, 30, 60, 100, 150, and 200 ms before the Test onset. The results of a two-way ANOVA (Hemisphere 2 X Prepulse 7) showed that the Prepulse was a significant factor for determining the amplitude of the Test response , inhibition was weaker than that of the shorter intervals in Experiment 1–1 with a significantly smaller response than the Test response only being observed for Prepulse 600 ms before the Test (p = 0.008) . This result suggests that the long-latency inhibition required a stronger prepulse. By considering the possibility that, in some subjects, the effects of a single click prepulse did not reach the threshold needed to exert an inhibitory action, we further tested a prepulse with a train of three clicks (Experiment 1–3). Although the train prepulse augmented inhibition and confirmed the time course of the long-latency inhibition  . No signWe next examined the short-latency inhibition in more detail using 10–70 ms Prepulses in a 10-ms step (Experiment 1–4). Inhibition was the greatest on average for the 40ms-Prepulse (27.7%), but did not differ significantly among the 20–70 ms-prepulses (p > 0.87). In both hemispheres, the time course of inhibition did not show a single peak , implyin5,60 = 0.85, p = 0.52) nor Hemisphere  significantly affected the amplitude of the Prepulse-evoked response in Experiment 1–1. Therefore, there was no clear relationship between the amplitude of the Prepulse-evoked response and %PPI . Similarly, the amplitude of the Prepulse-evoked response did not differ significantly among the seven prepulse conditions  or between hemispheres  in Experiment 1–4. The correlation coefficient r between the Prepulse-evoked amplitude and %PPI was 0.03 ± 0.48 for the left hemisphere and 0.21 ± 0.4 for the right.The peak latency and amplitude of the Test- and Prepulse-evoked responses are listed in Tables 4,48 = 35.08, p = 1.1 x 10−13, partial η2 = 0.75), but not Hemisphere  as a significant factor for the amplitude of the Test-evoked response. As shown in 3,36 = 11.16, p = 2.5 x 10−5, partial η2 = 0.48). When %PPI was plotted against the amplitude of the Prepulse-evoked response, their function was almost linear . On the other hand, as shown in 1,12 = 6.59, p = 0.025, partial η2 = 0.36). When the amplitude and %PPI of the Prepulse-evoked response for the 6030 Prepulse were plotted in Therefore, we compared two Prepulses, one with clicks at 60 and 50 ms (6050 Prepulse) and another at 60 and 30 ms (6030 Prepulse) before the Test onset . The resThe results of the present study showed that Change-N1m was clearly inhibited by a weak leading change stimulus and also that this inhibition reflected inhibitory actions on the generation of Change-N1m, but not an exclusive event within PC-PC excitatory synapses. Since Change-N1m used in the present study as the Test cortical response represents a kind of higher brain function to automatically detect changes in the sensory environment and to facilitate the execution of appropriate defensive behaviors , the preThe results showing that the Prepulse at approximately the excitatory threshold suppressed the Test response indicated that inhibition cannot be explained by passive events such as the refractory period or fatigue. Although a depressing PC-PC synapse is one candidate for the inhibitory mechanism, weak activation is unlikely to be the main mechanism regardless of whether it is presynaptic  or postsSince the Test and Prepulse have the same properties, they are expected to activate the same circuits. Therefore, low-threshold self-regulatory inhibition was considered to be a fundamental mechanism of control for the change-detection circuit in the present study. In order to evoke a clear MEG response such as Change-N1m in this study, a minimum of tens of thousands neurons have to be activated simultaneously . By consA-mediated IPSPs [in vivo whole-cell recording study in the mouse auditory cortex demonstrated that such a precise and stereotyped sequence of excitation and inhibition made the PC fire within a few milliseconds in response to auditory stimuli [Although non-invasive neuroimaging studies like the present study cannot reveal neural mechanisms in detail, some discussion can be made when the present results are compared with knowledge about interneurons obtained from whole-cell recording studies. Here we want to discuss the possible contribution of interneuron subclasses to the present PPI. Since the time course of inhibition was drawn using a weak prepulse of a single click at approximately the excitation threshold Figs  and 3A, ed IPSPs ,30,47–50ed IPSPs , 28 ms ied IPSPs , and ear stimuli . Previou stimuli , played  stimuli ). PV int stimuli , which a stimuli .Long-latency inhibition at approximately 600 ms differed from short-latency inhibition in late-onset, long duration, and weak inhibition. A candidate responsible for late inhibition is a somatostatin (SOM)-positive interneuron, which predominantly is a Martinotti cell ,56. Its SOM interneurons were previously reported to make unspecific dense connections with neighboring PCs, and thus, are considered to provide a ‘blanket of inhibition’  similar B-mediated inhibition [A and GABAB receptor-mediated inhibition by neurogliaform neurons [The present results implied the presence of a second short-latency inhibition 20–30 ms after the first one Figs  and 3E. hibition ,49, and  neurons . However neurons ,64. Ther neurons ,66, therThe present results showed that, in the Change-N1m circuit, excitation and inhibition always coexisted, thereby supporting the importance of interneurons to process sensory information. However, this also indicates the difficulties associated with human studies without methods such as voltage-clamping to observe these events separately. Even with such limitations, the present method appeared to be useful for evaluating inhibitory functions in each individual. Since the inhibitory system is similar at least in several respects across layers, cortical areas, and species (e.g. ,48,54,67


The presence or absence of a phosphorylation on a substrate at any particular point in time is a functional readout of the balance in activity between the regulatory kinase and the counteracting phosphatase. Understanding how stable or short-lived a phosphorylation site is required for fully appreciating the biological consequences of the phosphorylation. Our current understanding of kinases and their substrates is well established; however, the role phosphatases play is less understood. Therefore, we utilized a phosphatase dependent model of mitotic exit to identify potential substrates that are preferentially dephosphorylated. Using this method, we identified >16,000 phosphosites on >3300 unique proteins, and quantified the temporal phosphorylation changes that occur during early mitotic exit (McCloy et al., 2015  Specifications TableValue of the data1Phosphorylation is a dynamic modification, and therefore to fully understand the meaning of a specific phosphorylation, its half-life must be known. The stability is an output of the activity of the regulatory kinase and phosphatase A. In ord22.1In order to analyze temporal events during mitotic exit, highly synchronized cell cultures are needed. To achieve this, we utilized a two-step synchronization protocol using HeLa cells B. Briefl313C615N4-l-Arginine (Arg 10) and 13C615N2-l-Lysine (Lys 8) (Silantes GmBH), which was supplemented with 10% dialyzed FBS and 4 mM glutamine. To ensure complete labeling of >97%, cells were cultured for approximately six doublings in heavy or light media, with fresh media replaced every two days and sub-culturing performed when cells reached 90% confluence. After labeling, cells were synchronized as per HeLa cells were SILAC-labeled by culturing in DMEM where the natural “light” Lysine and Arginine were replaced by “heavy” isotope-labeled amino acids 42 phosphopeptide enrichment as previously described Cells were lysed in urea lysis buffer  phosphine (TCEP), 1 mM EDTA, 8 M urea and 20 mM HEPES), sonicated, and then iodoacetamide was added to 100 mM. Protein concentration was determined by the Bradford assay . Samples were then mixed 1:1 (light:heavy) based on quantification of total protein and digested in-solution with modified TPCK treated trypsin (Promega). Peptides were desalted on C18 solid-phase extraction columns and separated into 9 fractions by strong-cation exchange (SCX) using the ÄKTApurifier  followed by TiO5http://www.maxquant.org) using default settings with the inclusion of match between runs option. Peptides were assigned incorporating modified arginine-10 and lysine-6, with a maximum of 2 missed cleavages, using the fixed modification carboxyamidomethylation, and variable methionine oxidation and STY phosphorylation. Database searching was performed using the Andromeda search engine integrated into the MaxQuant environment ftp://ftp.uniprot.org). Precursor mass tolerance was set at 20 ppm for initial search, fragmentation peptide to 0.6 Da. To ensure high quality protein identifications, MS spectra were also searched against a reverse database of a similar size with the false discovery rate limited to <1%. Known contaminants identified by MaxQuant were filtered out of the initial dataset.Mass spectra were processed with version 1.2.7.4 of the MaxQuant software package  via the PRIDE partner repository http://prospector2.ucsf.edu) with the search key gsmtp1s5q7. In addition, a summary of this data, in an easy to use excel spreadsheet, is provided with this manuscript  was used to identify increased and dephosphorylated phosphopeptides, respectively. A moderated t-test was used to identify phosphosites that are significantly up or down-regulated using Linear Models for Microarray and RNA-Seq Data (LIMMA) package in R p.value>0.05), and had log2 ratios between −0.25 and +0.25 with a standard deviation <0.5. t-statistics, p-values and adjusted p-values for all phosphosites.To ensure that only highly confident protein identifications were reported, phosphosite identifications were filtered in excel for those with a localization probability greater than 0.75, a minimum MaxQuant score of 30 and a maximum posterior error probability of 1%. A fold change cut off of ≥4 (log8http://kinomexplorer.info) Annotation of upstream kinase was done using the reported minimal consensus motifs for each kinase 9z-sections were taken, de-convolved, and displayed as 2D maximum projections using Image J. False coloring and overlays were performed using Adobe Photoshop CC 2015 software.To validate the motifs observed in