The different conformations of WalK reveal the intrinsic dynamic properties of a sensor histidine kinase structure as a molecular basis for multifunctionality. Lactobacillus plantarum that shares 60% sequence identity with its homologue VicK from Streptococcus mutans, are presented. WalK adopts an asymmetrical closed structure in the presence of ATP or ADP, in which one of the CA domains is positioned close to the DHp domain, thus leading both the β- and γ-phosphates of ATP/ADP to form hydrogen bonds to the ∊- but not the δ-nitrogen of the phosphorylatable histidine in the DHp domain. In addition, the DHp domain in the ATP/ADP-bound state has a 25.7° asymmetrical helical bending coordinated with the repositioning of the CA domain; these processes are mutually exclusive and alternate in response to helicity changes that are possibly regulated by upstream signals. In the absence of ATP or ADP, however, WalK adopts a completely symmetric open structure with its DHp domain centred between two outward-reaching CA domains. In summary, these structures of WalK reveal the intrinsic dynamic properties of an SK structure as a molecular basis for multifunctionality.Two-component systems (TCSs) are key elements in bacterial signal transduction in response to environmental stresses. TCSs generally consist of sensor histidine kinases (SKs) and their cognate response regulators (RRs). Many SKs exhibit autokinase, phosphoryltransferase and phosphatase activities, which regulate RR activity through a phosphorylation and dephosphorylation cycle. However, how SKs perform different enzymatic activities is poorly understood. Here, several crystal structures of the minimal catalytic region of WalK, an essential SK from  The cells were harvested and resuspended in lysis buffer  and lysis was completed by sonication. The supernatant was collected after centrifugation at 40 000g and was incubated for 1 h at 4°C with 1 ml Ni2+–NTA resin (BBI life science) pre-equilibrated with the lysis buffer. After being washed with the lysis buffer, the protein on the beads was eluted with an imidazole gradient of 50–500 mM. The best fractions of the protein eluates were concentrated using a Centricon (Millipore) and loaded onto a Superdex 200 size-exclusion chromatography column  in a buffer consisting of 20 mM Tris–HCl pH 8.0, 100 mM NaCl, 5 mM β-mercaptoethanol. Peak fractions were collected and concentrated to 15 mg ml−1. Initial screening was set up using sitting-drop vapour diffusion with several commercial crystallization kits. The best protein crystals were found in 50 mM bis-tris pH 5.6, 1.0 M ammonium sulfate, 1% polyethylene glycol 4000. To obtain the ATP-bound state, 4 mM AMPPCP and 4 mM MgCl2 were added to the protein preparation before crystallization. Selenomethionine (SeMet)-labelled WalK was expressed in SeMet-substituted minimal M9 medium and was purified and crystallized as for the native protein  was amplified by PCR using 2.2.v/v) glycerol in mother-liquor solution]. Diffraction data sets were collected with a Quantum 315 detector at station BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, People’s Republic of China  and Coot  using NAMD 2.8  in Bio3D  and the CA domain (amino acids 271–450) were cloned in the pET-His vector, and the proteins were expressed and purified as described above. All lpWalK and smVicK samples were prepared in the same buffer consisting of 20 m3.3.1.a and Supplementary Fig. S1a). The γ-phosphate in the lpWalK–AMPPNP complex could not be defined, probably because of hydrolysis. However, the overall structure showed the same conformation as lpWalK co-crystallized with AMPPCP . Indeed, we also observed the same conformation for lpWalK that was crystallized in the presence of ADP (Table 1b).The catalytic region of lpWalK contains one DHp domain (amino acids 370–450) and one CA domain (amino acids 450–624). To obtain ATP-bound states, we crystallized the lpWalK fragment in the presence of the nonhydrolyzable ATP analogues AMPPCP and AMPPNP. Surprisingly, both structures resemble the smVicK catalytic region . The CA and DHp interactions are in cis rather than in trans. Residues Arg558 and Phe559 of the F box form a small interface between the DHp and open CA domains through hydrogen bonds and hydrophobic interactions, as observed in smVicK. These residues, together with the other interacting residues, including Thr573, Leu575, Ile579 and Glu582 from the CA domain and Phe385, Leu439, Leu442, Asp446 and Glu438 from the DHp domain, are highly conserved in WalK homologues. The buried surface area is as small as 682.8 Å2, but this interface appears to be further stabilized by a hydrogen-bond network consisting of Asp446, Arg558 and Glu582, probably as a result of subtle conformational changes in the F and G2 boxes induced by nucleotide binding.The overall conformations of these nucleotide-bound lpWalK structures represent an asymmetric closed state with one CA domain positioned close to the DHp domain, while the other CA domain reaches out, forming an open conformation Fig. 1b. The Cc). The conserved residues His391 and Glu392 in α1 form the first hydrogen-bond network with Gln506 and Asn510 in α4. Gln506 interacts with Glu392 through a hydrogen bond that is essential for His391 phosphorylation, as has been demonstrated in the chimeric EnvZ . These key residues in the closed DHp and CA interface are largely conserved between lpWalK and smVicK . Although Glu427 in lpWalK is substituted by Asp252 in smVicK, the hydrogen bond formed by the side chain to Tyr465 is preserved. Similarly, Met472 inlpWalK is substituted by Gln297 in smVicK, but the hydrophobic interaction formed between their side chains and Tyr403 is also preserved. Interestingly, the interaction between Lys476 and Ala406 in lpWalK is not found in smVicK as Lys476 is substituted by Gln301. Together, our data indicate that WalK adopts a closed structure in the presence of ATP analogues or ADP, which is characterized by the asymmetric positioning of the two CA domains; only one monomer of the dimer switches to a closed conformation while the other one remains open. The structures of the two CA domains are nearly identical (r.m.s.d. of 0.36–0.54 Å for 120 Cα atoms), except for a 15.3° rotation of α3 toward to the DHp domain in addition to the flexible loops L3 and L5 and the G1 box . The rotation of α3 avoids stereo clashes with the DHp domain when forming the closed conformation.The CA domain in the closed conformation makes a more extensive interface with the DHp domain Fig. 1c. The ccK Fig. 1d. Altho3.2.a, 2b and 2cd).In the closed lpWalK state, the ATP/ADP ligands are clearly defined . The α-phosphate (αP) forms hydrogen bonds to the amine of Asn514 and the main chain of residues 574–577 from the G2 box. The β-phosphate (βP) forms hydrogen bonds to Gly572 and Thr573 from the G2 box. Lys517 and Asn513 from α4 can both connect to the γ-phosphate (γP) through hydrogen bonds, while Lys517 and Tyr518 both form hydrogen bonds to βP in ADP-bound lpWalK , consistent with our previous finding that the mutation of homologous residues in VicK (Lys341A/Tyr342A) clearly reduces the autokinase activity . A tight hydrogen-bond network can be postulated between His391 ∊N and ATP γP, as indicated by alignment of the closed lpWalK state with the bsWalK CA structure bound by ATP . In the open conformation, however, these interaction partners are approximately 15–17 Å away, whereas His391 has two rotameric conformations that are fixed through its interaction with AMPPCP or ADP in the closed conformations . Together, our results suggest that ATP/ADP makes extensive interactions with the CA domain as well as with the phosphorylatable histidine in the closed conformation.The ATP phosphates are also involved in extensive interactions with lpWalK Fig. 2d. The αalK Fig. d, consiTP Fig. 2f. In thns Fig. 2c. Toget2+ is present, showed that the structures are nearly identical . Moreover, both nucleotides aligned except for the γP positions, which were 3.3 Å apart . Two coordinations by the hydroxyl groups of βP and γP are lost in our lpWalK structure bound to AMPPCP, which makes lpWalK unable to chelate Mg2+ stably.A magnesium ion was not identified in the ATP/ADP-binding pocket of the closed lpWalK structures. Our alignment of the closed CA domain with the bsWalK structure, where MgKd range 300–500 µM.We then measured the binding affinities of AMPPNP, ADP and AMP for the WalK proteins lpWalK and smVicK using isothermal titration calorimetry (ITC) experiments (Table 23.3.a). The CA domains reach out, causing the ATP-binding pocket to be positioned further away from the phosphorylatable histidine His391 in the DHp domain; we therefore defined this structure as representing the open state of WalK.We also determined the crystal structure of the catalytic region of lpWalK at 3.2 Å resolution in the absence of ATP/ADP. Unlike the closed lpWalK described above, the overall structure shows that the two CA domains symmetrically centre on the four-helical bundle of the DHp homodimer Fig. 3a. The CS. mutans VicK .The structures of both CA domains are similar to that of its close homologue c). The key residues in the open DHp and CA interface between lpWalK and smVicK are completely conserved . Although Glu438 is substituted by Asp263 in smVicK, the hydrophobic interaction is preserved as both have similar side chains. The buried surface area is barely 689.5 Å2, thus suggesting that this interface is not strong. Moreover, the key residues in the CA domain are located in the lid-covering loop of the ATP-binding pocket, including the F and G2 boxes. Indeed, a global alignment of lpWalK with the open chain of smVicK indicated an ∼4° rotation of the CA domain relative to the DHp . A dramatic rotation (∼46.1°) of the lpWalK CA domain was observed when it was aligned with T. maritima HK853 in the open state .A small interface is formed between residues Arg558, Phe559, Thr573 and Leu575 of the CA domain and Val389, Glu392, Met435, Leu439 and Asp446 of the DHp domain Fig. 3c. The ked Fig. 3d. Altho3.4.a). The monomer connecting the open CA domain adopts two straight helices, whereas the other monomer α1 bends towards the dimeric centre. When compared with their positions in open lpWalK, both DHp domains are well aligned except for the bent α1 . The bending of the α1 N-terminal tail is 25.7° at Pro396. Coordinately, the closed CA domain has a rotation of 57.7° and 9.1 Å difference along the DHp domain, while the open CA domain is positioned with a 15.8° difference . The asymmetrical structure of the DHp domain is comparable to that of smVicK, with nearly the same bending angle (r.m.s.d. of 0.78 Å for 59 Cα atoms). In the light of recent structural evidence from the SKs CpxA and DesK .To further understand the dynamics of the asymmetric helical bending in the DHp domain, we performed molecular-dynamics (MD) simulations. We observed that two straight α1 helices of the DHp domain from the open lpWalK were randomly bent by 16.7–26.8° during 50 ns of simulation, indicating that the bending is an intrinsic property of the α1 helices (Supplementary Movie S2). In contrast, the DHp domain from the closed lpWalK structure behaved completely differently (Supplementary Movie S3). The straight α1 helix became bent towards the central axis of the DHp domain, while the bent α1 helix retracted and became straight, thus resulting in switched α1 helical bending at ∼3 ns  and could not switch back to a straight conformation during the rest of the 35 ns of simulation. We reasoned that the resultant DHp structure might have unequal lengths of the α1 helices. To test this possibility, we simulated the DHp domain after removing the extra helical region (amino acids 372–383) to make the α1 termini of equal lengths. However, this DHp did not exhibit any switches during 20 ns of simulation . We then shifted the helical region (amino acids 375–383) from the bent α1 to the straightened α1 by using the simulated DHp structure in Fig. 5c) and continued the simulation under the same conditions. We observed that α1 successfully switched back in 15 ns . Finally, we tested whether the helical property of the extra helical region was required for this function. We rebuilt the helical region as a random coil during a 40 ns simulation. Although the random coil is positioned freely, the connected α1 did not bend at all (Supplementary Movie S5).Surprisingly, the DHp domain retained a bent α1 state Fig. 5c and coTogether, these MD studies revealed that the helical bending of the two α1 N-termini at the kink-residue proline is an intrinsic property of the homodimeric DHp domain, and the length and helicity of the α1 helix determine its helical bending capability. The α1 with a longer N-terminal helix and higher helicity appears to promote a favourable bending towards the central axis of the dimeric DHp domain. Moreover, the two α1 termini exhibit mutually exclusive bending and are able to switch sequentially.4.et al., 2009et al., 2014et al., 2014et al., 2016et al., 2012Lactobacillus.The ability of one SK to have both phosphorylation and dephosphorylation enzymatic activities is believed to rely on the flexibility of its conformation, such as that of DesK, HK853 and CpxA, as demonstrated in several structural studies . In addition, the γP of ATP was visible when lpWalK was co-crystallized with AMPPCP but not AMPPNP. Interestingly, although ATP is chemically unstable, its γP has been observed in several SK structures, including the bsWalK CA domain and CpxA . The open monomer of lpWalK has a small interface between the CA and DHp domains, which is also found in the open lpWalK structure . However, it is completely lost in the closed monomer of the closed lpWalK, whereas Arg558 switches its hydrogen bonds from Asp446 in α2 to Asn388 in α1 , whereas in the chimeric EnvZ the corresponding residue Asn347 interacts with an Mg2+ ion , and mutation of this residue disrupts the autokinase activity of smVicK  in one S. pneumoniae cell . Continuous simulation made the α1 helix switch back only when α1 contained an extended helix . These observations support a helix–coil transitional switch mechanism of the N-terminal α1 helices during autokinase activation  connects a transmembrane domain and allows the transmission of a variety of extracellular stimuli to downstream catalytic activities in many SKs (Dunin-Horkawicz & Lupas, 2010et al., 2006et al., 2012et al., 2006et al., 2012SK activities are largely modulated by upstream signals In summary, these observations suggest that the four-helical bundle of the DHp domain may have internally coordinated motions, which cooperate with the CA-domain repositioning and produce different conformations of WalK. ATP/ADP binding may modulate the dynamics of the CA domain. The dynamic properties of the DHp domain are likely to be essential for the enzymatic activities of WalK, which are largely modulated by upstream functional domains in response to a variety of stimulatory signals.Lactobacillus plantarum WalK, 4u7nPDB reference: bound to AMPPNP, 4u7oPDB reference: bound to ADP, 4zkiPDB reference: bound to AMPPCP, 5c93PDB reference: Click here for additional data file.10.1107/S2059798317013043/di5014sup2.movSupplementary Movie S1.. DOI: Click here for additional data file.10.1107/S2059798317013043/di5014sup3.movSupplementary Movie S2.. DOI: Click here for additional data file.10.1107/S2059798317013043/di5014sup4.movSupplementary Movie S3.. DOI: Click here for additional data file.10.1107/S2059798317013043/di5014sup5.movSupplementary Movie S4.. DOI: Click here for additional data file.10.1107/S2059798317013043/di5014sup6.movSupplementary Movie S5.. DOI: 10.1107/S2059798317013043/di5014sup1.pdfSupplementary Figures and Supplementary Movie captions.. 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Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the  Plants tightly control the balance between cell division and differentiation in order to maintain a specific number of dividing stem cells to sustain indeterminate growth and at the same time to provide enough cells for differentiation into appropriate tissues and organs5. In Arabidopsis, the CLAVATA3 (CLV3) peptide controls stem cell maintenance and organ specification in the SAM8. These CLV3 peptides are secreted from the outer cell layers of the SAM and act on the central domain depending on a variety of homo- and heteromultimeric complexes of plasma membrane-localized Leucine-Rich Repeats Receptor-Like Kinases (LRR-RLKs), including CLV1, BAM1 (BARELY ANY MERISTEM 1), BAM2, BAM3, CLV2, CRN (CORYNE) and RPK2 (RECEPTOR-LIKE PROTEIN KINASE 2)9 (and references therein). In the RAM of Arabidopsis, an analogous pathway fine tunes the balance between stem cell differentiation and maintenance10. CLE40 would act through the CLV1 and ARABIDOPSIS CRINKLY 4 (ACR4) receptors to regulate the Quiescent Center stem cell niche11.Plant growth relies on stem cells located in meristems from which most tissues and organs are post-embryonically derived e.g. the Shoot and Root Apical Meristems (SAM and RAM)12. Meanwhile, cell divisions are activated in cortical and pericycle cells13, leading to the formation of a nodule primordium that is colonized by infection threads containing rhizobia. The primordium then differentiates into a nodule in which bacteria fix atmospheric nitrogen into ammonium within the central nitrogen fixation zone, which is then assimilated by plant cells. In Medicago truncatula nodules, the apical meristem is continuously acting, leading to an indeterminate growth of the nodule, and is contiguous to a differentiation zone where rhizobial infections occur. The organization of the Nodule Apical Meristem (NAM) is therefore very similar to the RAM and SAM15, pointing out that a CLE/LRR-RLK may be involved in controlling the balance between cell division and differentiation in the nodule apex. Interestingly, MtCLE12 and MtCLE13 peptides were reported to be expressed in the apical region of the nodule16.When legume plants are deprived of nitrogen, nodule organs form on their root systems following a symbiotic interaction with nitrogen-fixing soil bacteria, collectively named rhizobia. The specific recognition between the two symbiotic partners allows bacteria to penetrate root hairs and progress towards the root cortex within infection threads17. Understanding the regulatory mechanisms that allow legume plants to adapt nodulation and nitrogen fixation rates to the balance between fluctuating nitrogen resources, in time and space, and the plant growth energy demand, may open perspectives to optimize plant productivity of legume crops. Such applied objective is key to further reduce nitrogen fertilizer inputs and their associated economic costs and environmental drawbacks, while maintaining, or even improving, legume yields under sustainable agricultural practices. The AON regulatory pathway involves a long-distance root-to-shoot-to-root systemic signaling allowing suppressing subsequent nodule initiation depending on soil nitrogen availability or nodule number. In different legumes including M. truncatula, the AON pathway was shown to rely on a CLE/LRR-RLK regulatory module, involving the production in rhizobia inoculated roots of peptides such as MtCLE12/MtCLE13 or LjCLE-RS1/LjCLE-RS2  that are proposed to move to shoots through the xylem vasculature and to act depending on the MtSUNN (Super Numeric Nodules)/LjHAR1 (Hypernodulation and Aberrant Root)/GmNARK (Nodule Autoregulation Receptor Kinase) subclass XI LRR-RLK receptor closely related to Arabidopsis CLV125. In Arabidopsis, CLE6 overexpression in roots affects shoot morphology26, indicating that CLE peptide-mediated long-distance signaling is not specific to legumes. Unlike the Arabidopsis clv1 mutant however, legume mutants affecting the protein most closely related to AtCLV1 do not show any SAM defect, suggesting a functional diversification among plants and/or different functional redundancies between LRR-RLKs. Other symbiotic AON mutants affected in LRR-RLKs were additionally identified, including CLV2-homologs from pea and Lotus, LjKLAVIER (KLV) that is closely related to the Arabidopsis RPK2 receptor, as well as the MtCRN kinase closely related to AtCRN28, suggesting that, similarly as for SAM and RAM, complexes of LRR-RLK are involved in the AON systemic signaling. Accordingly, MtSUNN was shown to form homo- and hetero-dimers with MtCLV2 and MtCRN28. In contrast to the nark/sunn/har1 AtCLV1-related mutants, MtCLV2- and MtRPK2-related mutants show SAM defects, indicating that partially overlapping receptor complexes controls SAM activity and nodule number in legumes29.As nodulation and nitrogen fixation are energy-consuming processes, a tight negative control of nodule number and activity is exerted by the plant depending on soil nitrogen availability and on the plant photosynthetic capacity to provide carbon required for ammonium assimilation, so-called “Autoregulation of Nodulation” (AON)30. Interestingly, opposite phenotypes were observed in the cra2 (compact root architecture 2) mutant which is affected in another subclass XI LRR-RLK31. In Arabidopsis, CEP peptides regulate root development depending on CEP receptors (CEPR1 and CEPR2) closely related to CRA234. Accordingly in M. truncatula, the CEP1 peptide requires CRA2 to regulate nodule and lateral root development35.More recently, another family of secreted peptides, referred to as CEPs , mostly produced under nitrogen-starvation, was shown to positively regulate nodulation and to inhibit lateral root formationM. truncatula receptors that are associated to the symbiotic nodulation based on their expression pattern. A single gene, MtNRLK1, was identified within the 10 proteins most closely related to SUNN as being upregulated by rhizobia. Interestingly, whereas the expression pattern of MtNRLK1 and MtSUNN is overlapping in tissues associated to the root vasculature, MtNRLK1 expression in the nodule apical region is, in contrast to MtSUNN, partially overlapping with the expression patterns of MtCLE12 and MtCLE13, leading to the hypothesis that a putative short-distance action of these peptides in the NAM might rely on the MtNRLK1 receptor. However, MtNRLK1 knock-down and ectopic expression did not reveal any significant nodulation phenotype, either considering nodule number, size, organization, nitrogen fixation capacity, or a requirement for the CLE long distance AON regulatory pathway.The objective of this work was to identify SUNN-related M. truncatula . For nine of the ten best hits, the expression pattern was available at the Medicago Gene Expression Atlas (MtGEA) (http://mtgea.noble.org/v3/) and amongst these, only one gene , showed a nodulation-enhanced expression profile and was therefore designated MtNRLK1 (for Nodule-induced RLK1).The full amino acid sequence of MtSUNN was blasted against the predicted proteome of M. truncatula , showing 82% of similarity and 73% of identity. Based on available RNAseq transcriptomic data36, the Medtr3g449390 gene is expressed more strongly in roots than in nodules  in a Sinorhizobium meliloti-induced nodulation kinetic  using Agrobacterium rhizogenes transformation. Two independent RNAi constructs were designed  insertional mutant affecting the MtNRLK1 genomic locus. The NF2218 line contains a Tnt1 insertion located in an exon corresponding to the region encoding the kinase domain of MtNRLK1  plants   Fig. . The nument Fig. . Again, MtNRLK1 and MtSUNN expression patterns overlap in root vascular tissues, we investigated whether MtNRLK1 could, like MtSUNN, be involved in the systemic AON regulation depending on the CLE12/13 peptides16. As previously observed in WT roots, a strong decrease in nodule number was detected in nrlk1 transgenic roots expressing a 35S:MtCLE13 construct compared to the 35S:GUS (control)  Fig. . This suM. truncatula nodulation. Amongst the 10 closest SUNN proteins, only MtNRLK1 showed a symbiosis-related expression pattern. MtNRLK1 is most closely related to the A. thaliana AtBAM3 receptor while MtSUNN is the closest M. truncatula homolog of AtCLV121. As both AtBAM3 and AtCLV1 function in SAM homeostasis by regulating stem cell identity42, we have made the hypothesis that MtNRLK1 might be involved in controlling the balance between cell proliferation and differentiation within the nodule potentially in relation with MtCLE12 and MtCLE13 peptides previously shown to be expressed in differentiated nodules16.The aim of this study was to identify and characterize subclass XI LRR-RLKs closely related to SUNN showing an enhanced expression during MtNRLK1 transcripts were detected at the base of nodule primordia, which accordingly to the nodule fate map proposed by Xiao et al.13 may correspond to cells derived from pericycle and inner cortex root layers, whereas MtCLE12 and MtCLE13 are expressed in whole primordia16. In addition, no pMtNRLK1:GUS activity was observed in infected root hairs, consistently with recent RNAseq analyses focused on the root epidermis or root hairs that revealed no expression of either MtNRLK1 or MtSUNN39. In differentiated nodules, MtNRLK1 is mostly expressed in the differentiation/rhizobial infection zone, in agreement with laser dissection transcriptomic datasets of Roux et al.36, which corresponds to cells derived from the nodule central meristem. This expression domain therefore partially overlaps with the expression pattern of MtCLE12 and MtCLE13 GUS transcriptional fusions reported by Mortier et al.16 in the nodule meristem and in the distal infection/differentiation zone. As in Arabidopsis CLE peptides are known to act as short distance signals that are perceived by neighboring cells46, the MtCLE12/MtCLE13 and MtNRLK1 expression domains could be consistent with a short-distance function of MtCLE12 and MtCLE13 peptides in the nodule apex.MtNRLK1 RNAi, mutant or overexpression lines. This suggests that MtNRLK1 may act redundantly with other RLKs still to be discovered. In Arabidopsis, bam3 single mutants also have a WT phenotype suggestive of a functional redundancy9. MtSUNN is most likely not a candidate RLK functionally redundant with MtNRLK1 in the nodule apex as no SUNN transcripts were detected in the nodule meristem or differentiation/rhizobial infection zones. In Arabidopsis, the expression pattern of AtBAM3 and AtCLV1 is also divergent, even though both receptors were associated to AtCLV3 peptide signaling in the SAM9. As MtNRLK1 is closely related to the Medtr3g449390 protein, a functional redundancy may exist between these two receptors despite the expression of this gene is not nodule-enhanced and even lower in nodules than in roots. As the MtSUNN expression pattern is redundant with MtNRLK1 in vascular tissues of the root stele, accordingly to the expression profile reported in Schnabel et al.37, and for HAR1 in L. japonicus and NARK in Glycine max47, a functionally redundant unknown function of these genes may exist locally in roots. Finally, no functional data currently supports a role of MtNRLK1 in relation to the SUNN-dependent AON systemic pathway inhibiting nodulation depending on CLE12/CLE13 peptides24, as a 35S:CLE13 overexpression strongly reduced nodule numbers in nrlk1 mutants similarly as in WT plants.No significant nodulation phenotype could be however identified in MtNRLK1 expression is nodule-enhanced and tightly associated to the nodule apex containing the meristem and the differentiation zone, we could not observe any related symbiotic phenotype when deregulating up or down MtNRLK1 expression. Future studies, including the functional analysis of additional LRR-RLKs expressed in nodules and the production of double mutants, notably between MtSUNN and MtNRLK1 as well as between Medtr3g449390 and MtNRLK1, may give more insights about possible local and/or systemic functions of MtNRLK1 in symbiotic nodulation.Altogether, whereas Medicago truncatula Gaertn. cv. Jemalong A17 seedlings were grown and inoculated as previously described48. A nrlk1 mutant (NF2218) was identified from the Tnt1 insertional mutant collection generated at the Noble Foundation . Genotyping of the homozygous mutants was achieved using the LTR4 primer TACCGTATCTCGGTGCTACA and the NF2218-R1 AGCAAGTCCAAAATCAGCAA primer to amplify the Tnt1 insertion, and the NF2218-F1 primer ATGGTGGTGAGAAACCTGGA and the NF2218-R2 primer TTTCCCATGCAACACTTCAC to amplify the NRLK1 locus. The Sinorhizobium meliloti strain Sm102151 was grown at 28 °C in a Yeast Extract Broth medium  and MtSUNN  was isolated from genomic DNA. The promoters were fused to the uidA gene in the pKm43GWRolD vector53. Primers used for amplification are listed in Table For M. truncatula plants were grown in vitro in square Petri dishes on a nitrogen-poor solution ‘i’ with Kalys agar54. For the nodulation kinetic, susceptible root zones were harvested before inoculation and at 4, 6, 8 and 10 dpi with Sm2011 from plants grown in pouches and watered with nitrogen-poor solution ‘i’ medium. Infection threads were visible from 4 dpi on, nodule primordia at 6 dpi, and young nodules at 8 and 10 dpi. Tissue was collected by visualizing the green fluorescent bacteria under a stereomicroscope MZFLII  equipped with a blue-light source and a Leica GFP Plus filter set . For the nrlk1 phenotype analysis, plants were grown in a mixture of perlite/sand (3/1) and watered with the nitrogen-poor solution ‘i’. Inoculation with Sm1021 was performed 7 days post-germination and nodules were scored 15 dpi. To evaluate the nodule nitrogen-fixation capacity and histology, a more efficient nitrogen-fixing strain was used, S. medicae WSM419. Sections were obtained using a vibratome (Leica VTS1200) on agarose 1% embedded nodules (3 weeks post-inoculation) and imaged with a Fluo AZ100 macroscope (Nikon). Acetylene reduction assays were used to evaluate the nitrogen fixation capacity, as previously described31.For quantitative RT-PCR (qRT-PCR) analyses, Medicago truncatula Proteome v4 database using the BLASTP algorithm (http://www.jcvi.org/medicago/). In addition, the 10 proteins most closely related to MtNRLK1 in A. thaliana were retrieved from the TAIR10 protein database (https://www.arabidopsis.org/) by using the BLASTP algorithm. The MtCRA2 LRR-RLK, previously functionally characterized in M. truncatula regarding symbiotic nodulation, was also included. To generate a similarity tree using the SeaView version 4 program55, complete amino acid sequences of MtSUNN and MtNRLK1 as well as the A. thaliana and M. truncatula closest related sequences previously identified were used to generate a multiple alignment using MUSCLE56. Poorly aligned positions and divergent regions were eliminated with the G-blocks software57. A maximum-likelihood phylogenetic tree was obtained using the PhyML program58 as well as an aLRT (approximate Likelihood-Ratio Test) test for branch support.To identify MtSUNN-related LRR-RLKs, the full amino acid sequence of MtSUNN was blasted on the 4 acetate (5 M) precipitation, quality controlled, and quantified with a Nanodrop spectrophotometer . RNA (2 μg) was used for cDNA synthesis with the Superscript Reverse Transcriptase Kit . The samples were diluted 50 times and the qRT-PCR experiments were performed on a LightCycler 480  using the LightCycler480 SYBR Green I Master Kit according to manufacturer’s instructions (Roche). All reactions were performed in triplicate and averaged. The relative expression was normalized against the reference genes Histone 3-Like  or a 40S Ribosomal S8 Protein . Primers used are indicated in Table Total RNA was isolated with the RNeasy Plant mini kit  according to the manufacturer’s instructions. After a DNAse treatment, samples were purified through NHet al.59. Approximately 48 h after germination, radicles of WT or nrlk1 seedlings were sectioned 5 mm above the root tip with a sterile scalpel. Sectioned seedlings were infected by coating the freshly cut surface with an A. rhizogenes Arqua1 strain containing the vector of interest (CLE13 OE16). The A. rhizogenes strain was grown at 28 °C for two days on a solid agar-YEB medium supplemented with spectinomycin (100 µg/ml)60. Infected seedlings were grown on a Fahraeus medium supplemented with 1 mM NH4NO3 and kanamycin  in square Petri dishes for one week at 20 °C, 16-h photoperiod (150 μE m−2 s−1) then for one more week at 24 °C under identical light conditions. Subsequently, composite plants selected on kanamycin were transferred on a brown growth paper  on a Fahraeus medium without nitrogen, grown at 24 °C under identical light conditions for five days, and then inoculated with S. meliloti Sm1021 strain at OD600nm = 0.05.The protocol was adapted from Boisson-Dernier d-glucuronic acid as a substrate. Roots and nodules were vacuum infiltrated during 20 min and subsequently incubated in a GUS buffer at 37 °C. Incubation lasted 6 h and 10 h for pMtNRLK1-GUS and pMtSUNN-GUS, respectively. After staining, roots and root nodules were fixed, dehydrated, embedded with Technovit 7100 , according to the manufacturer’s instructions, and sectioned with a microtome . Three μm thick sections were mounted on coated slides . For tissue counter staining, sections were treated with a 0.05% (w/v) ruthenium red solution , washed in distilled water, and dried. Sections were mounted with Depex , and pictures were taken with a Diaplan microscope equipped with bright- and dark-field optics .GUS activity in transformed roots and nodules was analyzed using 5-bromo-4-chloro-3-indolyl-β-62. A 35S-labeled antisense probe, corresponding to a region spanning the open reading frame and the 3′ UTR of MtNRLK1, was produced using the T7 RNA polymerase (Invitrogen) on a PCR-amplified probe  on paraffin-embedded nodules were hybridized as previously describedNon-parametric tests were used to assess significant differences: a Mann and Whitney test when two conditions are compared and a Kruskal-Wallis test when more than two conditions are considered.Supplementary Information


DLC1; also known as STARD12 or ARHGAP7). DLC1 encodes a Rho-GAP that negatively regulates Rho by converting active GTP-bound Rho to inactive GDP-bound Rho, is associated with focal adhesions, and is down-regulated in many tumor types.Rho GTPases are key transducers of several fundamental cellular processes such as cell division, adhesion, migration, polarity, cytoskeletal remodeling, and apoptosis . They acDLC1 gene, which is located at chromosome 8p21-22, is a 1091 amino acid protein that contains three well recognized domains: an N-terminal sterile alpha motif (SAM) domain, a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain, and the Rho-GAP domain, located upstream from the START domain [DLC2 (STARD13), and DLC3 (KIAA0189 or STARD8) have also been identified. Although all three members of the family are widely expressed in normal tissues, only DLC1 knockout mice are embryonic lethal, while DLC2 and DLC3 knockout mice are viable, which suggests DLC1 is essential for embryonic development. In cancer, downregulation of DLC1 is also more important than DLC2 and DLC3 [DLC1-deficient mouse embryos have shown significant alterations in the cytoskeleton organization of actin filaments and focal adhesions, suggesting a crucial role for DLC1 in these processes in normal cells [The most abundant translation product of the human T domain . A SerinT domain . Two othand DLC3 . Fibroblin vivo and in vitro. In addition to its growth suppressing activities, DLC1 has other physiological functions in a variety of normal and transformed cell lines. The most prominent of these is its Rho-GAP activity, which strongly inactivates RhoA, RhoB, and RhoC, weakly inactivates Cdc42, and does not affect Rac1. The catalytic activity of DLC1 is mediated by a conserved “arginine finger,” and mutating this Arginine to Alanine abolishes the Rho-GAP activity of DLC1. Other key functions of DLC1 include its localization to focal adhesions, and the binding of several functionally important proteins such as tensin, which binds to sequences in the LR, and talin and focal adhesion kinase (FAK), which bind to distinct LR sequences located downstream from those that mediate tensin binding. The binding of tensin, talin, and FAK facilitates the association of DLC1 with focal adhesions. During early cell spreading, DLC1 is preferentially localized at the inner/mature focal adhesions, whereas phosphorylated paxillin occupies the outer/nascent focal adhesions. In addition, DLC1 down-regulates paxillin turnover, and does not require its Rho-GAP activity for this function. Instead, it requires the presence of FAK. Both DLC1 and FAK are key regulators of focal adhesion assembly/disassembly that also regulate paxillin turnover at focal adhesions [DLC1 is a bona fide tumor suppressor that can be down-regulated by gene deletion, point mutation, epigenetic promoter methylation, or post-translational mechanisms in a variety of human cancers, including malignancies of the lung, breast, colon/rectum, prostate, and liver. Restoration of DLC1 expression can inhibits tumor growth and suppresses cell migration We have recently determined that direct phosphorylation of four specific Serines in the DLC1 LR by the CDK5 kinase, a serine/threonine kinase, is a common mechanism by which the various functions of DLC1 can be activated . The CDK


Recently, cobalt phosphide (CoP) and molybdenum phosphide (MoP) received great attention owing to their superior catalytic activity and stability towards the hydrogen evolution reaction (HER) which rivals platinum catalysts. In this study, we synthesize and study a series of catalysts based on hybrids of CoP and MoP with different Co/Mo ratio. The HER activity shows a volcano shape and reaches a maximum for Co/Mo = 1. Tafel analysis indicates a change in the dominating step of Volmer–Hyrovský mechanism. Interestingly, X-ray diffraction patterns confirmed a major ternary interstitial hexagonal CoMoP Bulk hexagonal molybdenum phosphide (MoP) has shown stable HER activity in acidic solution with overpotentials of 140–246 mV for driving a current density of 10 mA cm−2 [et al. [0.6Mo1.4N2 allows the Co to tune the electronic states of molybdenum at the catalyst surface without disrupting the catalytic activity. Staszak-Jirkovský et al. [n+ cations in a CoMoSx chalcogel structure helped accelerate the rate-determining Volmer step. Ternary Chevrel-phase NiMo3S4 was reported by Jiang et al. [6S8]2 cluster units allow faster charge transfer.Transition metal-based pnictides ,9 2.6H2O, Acros), ammonium molybdate ((NH4)6Mo7O24.4H2O, Aldrich) and ammonium dihydrogen phosphate . A typical procedure is shown as follows. For preparing Co0.5Mo0.5P catalyst, 10 µmol ammonium molybdate, 70 µmol cobalt nitrate and 140 µmol ammonium dihydrogen phosphate were mixed in 50 ml water and were ultrasonicated until all salts were dissolved. The ratio of cobalt nitrate to ammonium molybdate was varied to create molar ratios [Co]/[Mo] of 0.1/0.9, 0.3/0/7, 0.5/0.5, 0.7/0.3 and 0.9/0.1. After mixing, the transparent solution was dried at 80°C in an oven. The solid mixture was then annealed in a quartz tube furnace with a 100 sccm Ar flow from ambient to 350°C at a rate of 10°C min−1 and then held at 300°C for 1 h. Then the gas flow was switched to H2/Ar mix flow . The temperature was increased to 800°C at a ramping rate of 15°C min−1 and held at 800°C for 2 h. Bulk CoP and MoP were prepared by the same procedure using corresponding precursors with [Co or Mo]/[P] = 1. To study the effect of annealing temperature, the precursors of Co0.5Mo0.5P were treated at 650°C and 1000°C under H2/Ar mix flow for 2 h after the annealing at 350°C under Ar.Hybrids of cobalt phosphide and molybdenum phosphide were prepared by a two-step thermal treatment of mixtures of cobalt nitrate (Co(NO2.2.φMoτP hybrids were verified by X-ray diffraction (XRD) patterns with a Bruker D2 PHASER diffractometer. X-ray photoelectron spectra (XPS) were collected on a VG Scientific ESCALAB 250 and the binding energy was calibrated using the C 1s peak at 284.6 eV.The micromorphology of hybrid catalysts were observed on a field emission scanning electron microscope (JOEL JSM−6700F) and on a JEOL JEM-2100 transmission electron microscope. The crystalline compositions of the Co2.3.2 active area was fabricated by drop-casting the catalyst ink on with a micropipette. The catalyst loading was 0.4 mg catalyst cm−2. The electrode was then dried under vacuum at room temperature. The electrolytes for electrochemical measurements were prepared with perchloric acid (Aldrich) and Milli-Q water (Millipore). Pt foil (purity 99.999%) was purchased from Aldrich. All the electrochemical measurements were performed in 0.1 M HClO4 (aq) electrolyte, which was deaerated with hydrogen gas before use. A platinum wire, and an Ag/AgCl reference electrode (3 M KCl) were used as the counter electrode and reference electrode, respectively. All potentials, E, are quoted with respect to reversible hydrogen electrode (RHE). The electrocatalysis was studied using linear sweeping voltage in the range of +0.2 V to −0.5 V (versus RHE). Electrochemical impedance spectroscopy (EIS) was performed in potentiostatic mode at an applied overpotential of 100 mV with frequency from 10 mHz to 0.1 MHz and amplitude of 5 mV. The accelerated deterioration test of electrocatalysts was examined by applying cyclic voltammetry in the potential range from +0.1 to −0.5 V versus RHE for 1000 cycles.Electrochemical measurements were performed in a three-electrode electrochemical cell using a Zahner ZENNIUM E electrochemical workstation. The electrodes for the electrochemical measurements were fabricated as follows. Catalyst ink was prepared by mixing catalyst powder with milli-Q water solution . In total, 5% Nafion dispersion (DuPont) was added  to the catalyst slurry. Catalyst coating on glassy carbon electrode with 0.196 cm3.φ . CoφMoτP (e.g. Co0.1Mo0.9P) is defined as the sample code of the obtained catalysts; it does not represent any crystalline phase. The CoφMoτP catalysts investigated were Co0.1Mo0.9P, Co0.3Mo0.7P, Co0.5Mo0.5P, Co0.7Mo0.3P and Co0.9Mo0.1P as shown in The goal of this investigation is combining Mo with Co in order to tune the properties of their phosphides. Early attempts found enhancement of the HER activity on MoP by the doping of small amounts of Co onto its hexagonal structure . The synφMoτP catalysts, MoP (the same process for making CoφMoτP) and CoP in 0.1 M HClO4 solution using a typical three-electrode electrochemical cell. The HER activity is compared using the overpotential (η) at 10 mA cm−2 of cathodic current density (η10) and the current density at η = 200 mV. For assessing the electrochemical analysis, in figure 1a, a Pt foil exhibits HER activity with η10 of 50 mV which is comparable to other studies. The polarization curve recorded with CoφMoτP showed good activity for the HER. It is observed clearly in the electrochemical cell that the hydrogen bubbled more vigorously with increasing overpotentials. The η10 of the MoP and the CoP catalysts were read at 250 mV and 283 mV, respectively, which are similar to the overpotentials reported previously ). Figure 5b shows the dependence of the obtained Tafel slopes on the cobalt content, φ. For Pt foil, the Tafel slope of 30.1 mV dec−1 suggests a Volmer–Tafel mechanism that the recombination of two Hads is rate-determining. The data in figure 5b showed decreasing Tafel slopes from 84.0 to 60.5 mV dec−1 for samples from φ = 0 to 0.5. These results suggest that at a small φ  hydrogen evolution occurs via a Volmer–Heyrovský mechanism in which slow adsorption of proton dominates the kinetics. For φ = 0.5, the small Tafel slope of 60.5 mV dec−1 indicates that the electrochemical desorption of hydrogen influences the reaction kinetics through the Volmer–Heyrovský mechanism much more than the case of MoP catalyst. Comparing the electrochemical properties of Co0.5Mo0.5P catalyst to bulk CoP and MoP catalysts, the small overpotential and the low Tafel slope of Co0.5Mo0.5P demonstrate that the presence of h-CoMoP2 crystal phase in the catalyst favours proton adsorption kinetics. To further ensure that h-CoMoP2 crystal phase enhances HER activity, we collected polarization curves from three 50/50 mixtures of CoP/MoP powder. The obtained polarization curves of the physical mixtures  showed HER activity ranging between pure CoP and MoP catalysts. This result indicates clearly that the physical mixture of CoP and MoP powder does not enhance the HER activity, while the h-CoMoP2 crystal phase does.The dependence of the gradient of logarithm of current density versus potential  corresponds to the reaction mechanism of the HER. The Tafel curves recorded on the Co0.5Mo0.5P, CoP and MoP samples as shown in Rs, is a sum of all ohmic resistances that the electron passed through from wires to the catalyst surface. The double-layer capacitance, Cd1, in parallel with the charge transfer resistance, Rct, contributes to the charge-transfer process; the capacitance of the catalyst coating, Cd2, in parallel with the resistance of ion conducting paths that develops in the porous surface, Rp. The curves in Rct. The Rct of the Co0.5Mo0.5P catalyst (92.0 Ω) is found much lower than CoP (154.1 Ω) and MoP catalysts (107.5 Ω). As reported recently, the Rct of bulk MoS2 was found to be 150 Ω at η = 150 mV [2/RGO has an Rct of 250 Ω at η = 120 mV [2C nanowires showed a low Rct of 90 Ω at η = 150 mV [0.5Mo0.5P catalyst is regarded as a catalyst with efficient charge transfer property. Efficient charge transfer kinetics reflects to the accelerating of the proton discharge step in the Volmer–Heyrovský mechanism and thus lowers its Tafel slope as aforementioned. The enhancement in Rct can be ascribed to the modification of d-band structure owing to the formation of Co–P–Mo linkage.We carried out EIS analyses on these catalysts to study the charge transfer in the catalytic turnover. The measurements were conducted at overvoltage of 100 mV. The Nyquist plots of the Co= 150 mV . MoS2/RG= 120 mV ; Mo2C na= 150 mV . Thus, t0.5Mo0.5P electrodes, we performed the accelerated deterioration experiment by sweeping the applied potential from −0.5 to + 0.2 V versus RHE in 0.1 M HClO4 solution. After 1000 cycles on the Co0.5Mo0.5P electrode, only a slight shift (11 mV increase in η10) in the polarization curve is observed as shown in figure 7a, indicating a good stability of the Co0.5Mo0.5P catalyst in the operating conditions. Faradaic efficiency was determined by performing a controlled potential electrolysis experiment using an H-type cell. The amount of hydrogen gas produced was calculated from the volume of gas at a fixed cathodic current density of 10 mA cm−2 in 0.1 M HClO4 solution for 60 min. The experimentally determined hydrogen quantity was compared to the calculated amount based on the charge consumed as shown in figure 7b. The Faradaic efficiency reached 98% after 60 min of operation. The cathodic current density recorded for driving the HER at overpotential of 165 mV as a function of time is plotted in the electronic supplementary material, figure S8. The current density slightly decreased about 5% after 6 h of electrolysis. These results imply that the Co0.5Mo0.5P catalyst is a highly efficient and cathodically stable HER catalyst in the acidic environment.To assess the stability of the Co4.φMoτP hybrids and their activity in the HER. The HER activity of the CoφMoτP hybrids showed a volcano shape with a maxima at Co content of 0.5. The Co0.5Mo0.5P catalyst possesses a smaller overpotential (165 mV for driving 10 mA cm−2 of current density) when compared with CoP and MoP and is comparable to bulk non-precious catalysts reported. The hexagonal CoMoP2 phase was found to be responsible for the HER activity in the Co0.5Mo0.5P catalyst. The Tafel analysis showed a change in the dominating step in Volmer–Heyrovský mechanism. The low Tafel slope of the Co0.5Mo0.5P catalyst suggests that the electrochemical desorption of adsorbed hydrogen is the rate-determining step in the HER. The Co0.5Mo0.5P catalyst showed low charge transfer resistance, high stability and high Faradaic efficiency; all demonstrate that the Co0.5Mo0.5P catalyst is a potential, high-performance electrocatalyst for water electrolysis in acidic environments.We have studied the relationship between the composition of Co


B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release , the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.Neuromuscular preparations exposed to Bothrops snakes are close to 74% according to the National System of Accidents Notification (SINAN) [2 contained in their venoms. One dominant effect of Bothrops snakebites is marked myotoxicity without any clinical sign of neurotoxicity. However, in vitro studies in amphibian, avian, and mammalian nerve-muscle preparations have shown neurotoxicity, with total and/or irreversible neuromuscular blockade, at very low concentrations of venom [2 (PLA2) myotoxins plus an indirect action caused by tissue anoxia has key roles in the effects of Bothrops venom on the muscle and nerve fibres, including immediate destruction of motor axons and complete depletion of intramuscular motor nerve trunks [In Brazil, the accidents caused by (SINAN) ; they co (SINAN) . General (SINAN) –5 and sy (SINAN) . Such efof venom  or toxinof venom –10 from e trunks . Bothrops genus are close to 50. Geographically, same species can be distributed all over the country while others are endemic, for example, Bothrops marajoensis, which is only found in the Marajó Island, Pará State, in the very North of Brazil [Brazilian species of thef Brazil , 13  which occurs concomitantly with twitch tension facilitation followed by presynaptic neuromuscular paralysis but without evidences of muscle damage [In neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND), concentrations as low as 1 or 5 e damage .2 (PLA2) have been isolated from B. marajoensis venom: Bmaj-9 (13679.33 Da) [2s [Three basic Asp-49 phospholipases A9.33 Da) , BmjeTX- Da) [2s  and were Da) [2s .Venom's toxins that act on presynaptic sites interfere in complex machinery where proteins, such as synaptophysin, synaptobrevin, and SNAP25, undertake specific roles at precise steps of neurotransmitter release. Synaptophysin, an integral protein of the synaptic vesicle, binds to synaptobrevin and may act to prevent vesicle docking at the axolemma of the terminal bouton; SNAP25 is a plasma membrane protein (t-SNARE), which in association with synaptobrevin forms a stable complex responsible for vesicle docking, priming, and fusion . Alterat in vitro to two major toxins isolated from the venom of B. marajoensis. Our hypothesis is that BmjeTX-I and BmjeTX-II affect neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.In this communication we describe our observations on the cellular pathology of the motor nerve terminal and skeletal muscle fibres exposedMale HY-line W36 chicks (4–8-day-old) were supplied by Globo Aves Agrovicultura Ltda . Animals were housed at 25°C under a 12 h light/dark cycle with free access to food and water. All procedures were approved by the Institutional Committee for Ethics in Animal Use  and are in accordance with the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA) guidelines. B. marajoensis snake venom were purified as described in full detail by Ponce-Soto et al. [BmjeTX-I and BmjeTX-II isolated fromo et al. .All reagents were obtained from Sigma-Aldrich  if not stated otherwise. Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH-FL335) was from Santa Cruz Biotechnology ; OCT-Tissue Tek was from Sakura Finetek. EPON EMBed-812 Kit, uranyl acetate, osmium tetroxide, lead citrate, and glutaraldehyde were from Electron Microscopy Sciences . Western blotting reagents were purchased from Sigma-Aldrich, Biorad, Amresco, and Kodak. All salts for the physiological solution were of analytical or sequencing grade.n = 12) were killed by halothane inhalation and immediately the biventer cervicis muscles were removed and mounted under a tension of 1 g in a 5 mL organ bath containing Krebs solution , pH 7.5 at 37°C, and carbogen aeration  [2 toxins (10 μg/mL). Such concentration was the same used by Ponce-Soto et al. [Chicks (μM) and potassium chloride  were obtained in the absence of field stimulation prior to toxins addition and by the end of the experiment (120 min).A bipolar platinum ring electrode was placed around the muscle and coupled to a Grass S48 stimulator . Isometric muscle contractions and contractures were recorded via a force displacement transducer  coupled to a physiograph . Contractures to exogenous acetylcholine , maintained in carbogen-aerated Krebs solution (control) or incubated with 10 μg/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, permeabilized in ethanol and methanol , rinsed with distilled water, and stained with haematoxylin and eosin (HE) for histological analyses.Longitudinal cryosections of CBC (n = 4), maintained in aerated Krebs solution (control) or incubated with 10 μg/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were pinned side by side to wax under slight longitudinal tension, immersion-fixed in Karnovsky's solution for 30 minutes at room temperature and overnight at 4°C. Afterwards, the samples were washed in 0.1 M sodium cacodylate buffer, post-fixed in 1% OsO4 solution for 2 h, washed in the same buffer followed by distilled water, contrasted with 5% uranyl acetate for 1 h, washed in distilled water, dehydrated in acetone series, and embedded in Epon resin . Ultrathin sections (60 nm thick) stained with uranyl acetate followed by lead citrate were examined in a Leo 906 transmission electron microscope . The ultrastructure of the muscle fibres and neuromuscular junctions and morphometry of synaptic vesicles were provided.Samples containing end-plate regions of CBC , maintained in carbogen-aerated Krebs solution (control) or incubated with 10 μg/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, permeabilized in ethanol and methanol , and in 0.1% Triton X-100 in phosphate buffered saline (PBS) , rinsed with PBS, and incubated overnight in a moist chamber at 4°C with appropriate primary antibodies . Control of the immunoreaction was done by omitting the primary antibodies. On the following day, the slides were allowed to return to room temperature, washed in PBS, and incubated with the appropriate FITC-conjugated secondary antibody  or TRITC-conjugated α-BgTX  for 2 h at room temperature and then mounted in glycerin jelly. The positive immunolabelling was accessed, using a fluorescence BX51TF microscope , from 6 serial sections and the positive reactions were counted for statistical purposes (morphometry).Transversal cryosections of CBC (n = 4), maintained in carbogen-aerated Krebs solution (control) or incubated with 10 μg/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were homogenized in 1 mL of antiprotease cocktail. Aliquots of 15 μg proteins were used for 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis . After electrophoretic proteins transfer to nitrocellulose membrane , the samples were blocked overnight at 4°C in PBS containing 5% w/v dried milk and probed with primary antibodies  for 4 h. Blots were washed in PBS and incubated with a corresponding HRP-conjugated secondary antibody diluted in PBS containing 1% w/v dried milk to a final concentration of 1/1000 goat anti-rabbit, 1 : 5000 rabbit anti-mouse, and 1 : 3000 rabbit anti-goat, respectively.CBC nerve-muscle preparation . Densitometric data of endogenous control were generated by incubating blots with GAPDH . After rinsing in PBS, the immunoreactive bands were detected by chemiluminescence  using X-ray film . Experimental data were expressed in terms of relative optical density. post hoc test with p < 0.05 indicating significance. All analyses were done using Prism software .Quantitative data were expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA followed by the Bonferroniμg/mL) displayed total and irreversible neuromuscular blockade after 31.2 ± 3.5 min and 30 ± 8.1 min (p < 0.05 from Krebs), respectively, similarly to previous report [2 toxin isoforms resulted from increase in the twitch response of indirectly stimulated neuromuscular preparations. None of the toxins interfered significantly with the contractures to exogenously applied ACh and KCl after 120 min incubation (n = 12) (CBC preparations incubated with BmjeTX-I or BmjeTX-II (10 s report . The neu(n = 12) .Light microscopy showed that control CBC (incubated for 120 min in Krebs solution) presented muscle fibres with normal morphology while CBC incubated with BmjeTX-I or BmjeTX-II presented regions with fibres with different pathologic states, including vacuolated or swollen fibres, presenting loosely and/or densely clumping of myofibrils  but alsoTransmission electron microscopy was used to assess whether BmjeTX-I and BmjeTX-II affect the subcellular integrity of muscle fibres and neuromuscular junction (NMJ) of biventer cervicis. Control muscles showed typical ultrastructure with paralleled myofibrils and organized sarcomeres. Longitudinal- and cross-sectioned myofibrils were typically separated by profiles of sarcoplasmic reticulum; subsarcolemmal nuclei and mitochondria were observed without any damage. Intramuscular axons showed well organized myelin sheath and normal neurofilaments and mitochondria Figures .Opposed to controls, CBC preparations incubated either with BmjeTX-I or BmjeTX-II presented hypercontracted fibres and clusters of swollen mitochondria and/or deprived of cristae, blurred Z line with loss of myofilaments, and sarcomeres disorganization. Diffuse swelling of the sarcotubular system was also observed and some fibres exhibited myonuclei with chromatin and nucleoli alterations. Intramuscular motor nerve fibres displayed multishaped alterations of the myelin sheath and axons Figures . Regions2 caused a presynaptic neuromuscular blockade, we further investigated the neuromuscular junction (NMJ) ultrastructure by TEM. Control samples incubated with Krebs solution showed no abnormal pre- and postsynaptic ultrastructure. The terminal bouton was clearly defined by a continuous axolemma; it was adjusted into a well-delineated synaptic gutter and covered by processes of the Schwann cell. As usual, the synaptic vesicles and mitochondria were polarized to presynaptic axolemma and Schwann cell, respectively; typically, the postsynaptic sarcolemma was unfolded in chick muscle; densities along its length were due to intramembranous presence of postsynaptic receptors (In order to support the twitch tension findings which showed that BmjeTX-I and BmjeTX-II PLAeceptors .p < 0.05) reduction in the density of synaptic vesicles inside the terminal bouton while the remaining vesicles were clumped together in small aggregates; a massive mitochondrial damage and absence of presynaptic membrane were also observed  , were well preserved for all experimental groups (data not shown).The proteins examined showed a 100% basal expression in control, whereas they showed a scale proportion where synaptophysin > SNAP25 > synaptobrevin in preparations incubated with BmjeTX-I and synaptophysin ≥ synaptobrevin = SNAP25 in preparations incubated with BjmeTX-II . The AChp < 0.01) were observed between control and PLA2s-incubated preparations  action was primarily at the motor nerve terminal, as suggested elsewhere [2 from B. marajoensis venom, likewise induced a total and irreversible blockade after 70 ± 5 min at same concentration and same nerve-muscle preparation; likewise, the contracture evoked by KCl and ACh remained unchanged by the Bmaj-9 [Chick biventer cervicis (CBC) preparation incubated with either of the toxins showed irreversible twitch tension blockade. The KCl- and ACh-evoked contracture was unaffected by either of the PLAlsewhere . Bmaj-9,e Bmaj-9 .μg/mL low concentration of B. marajoensis venom was able to induce neuromuscular blockade without depressing the responses to exogenous ACh and KCl, blocking postsynaptic acetylcholine receptors or interfering with the muscle contractile mechanisms [μg/mL produced total blockade at around 100 min [In agreement, 1 chanisms . Neverth 100 min  and prom2 toxins.Based on the data, it can be concluded that the presynaptic action of the venom could be on account of Bmaj-9 , BmjeTX-α-bungarotoxin labelling even in treated preparations (figure not shown) confirmed the normal and functional pattern of the postsynaptic machinery in controls and postsynaptic intactness in treated preparations (not shown).The blockade of the twitch tension in CBC preparations incubated with BmjeTX-I or BmjeTX-II was found to occur in connection with the shift of synaptophysin, synaptobrevin, and SNAP25 proteins from the biventer cervicis end-plates relative to the control counterparts which have shown total expression of the three proteins (IHC and WB data). In control CBC incubated with Krebs solution the immunolabelling of presynaptic proteins delineated their distribution in the synaptic contacts over the myofibres indicating an operative biventer cervicis contractile structure which was responsive to the indirect electric nerve stimulation; these data and the colabelling of acetylcholine receptors (AChR) as proved by Complex presynaptic machinery is necessary to release the neurotransmitter at the synaptic cleft of the neuromuscular junction. Initially the synaptic vesicles (SV) are attached to a fine, filamentous actin cytoskeletal network in the presynaptic portion of the nerve terminal. The neurotransmitter vesicle binds to an active target zone near calcium channels (tethering) and docks to the presynaptic membrane and an ATPase-dependent process facilitates the SV priming. Once primed the SV can fuse and release the neurotransmitter. This process is mediated by vesicle-associated, cytosolic, and membranous proteins .2 toxins. In contrast, the number of synaptic vesicles relative to the area of the terminal was significantly reduced in preparations incubated with Bjme-TX-II but not with Bjme-TX-I. Synaptophysin expression is related to SV integrity [Another important finding of this study was that the area of the terminals was significantly reduced in preparations incubated with Bjme-TX-I and Bjme-TX-II with reduction significantly higher with the former than with the latter. Moreover, the number of synaptic vesicles per terminal was significantly decreased when CBC preparations were incubated with either of the PLAntegrity  and its 2 neurotoxins (SPANs) paralyze the neuromuscular junctions (NMJs) in vertebrate skeletal muscles by reducing the content of SV. Studies using primary neuronal cultures show synaptic swelling, with surface exposure of the luminal domain of the synaptic vesicle protein synaptotagmin I, and exocytosis of neurotransmitters. Other studies using an equimolar mixture of lysophospholipids and fatty acids, which mimics the biological effects of SPANs, indicate a possible role of local lipid changes in SV release [Snake presynaptic PLA release .μg/mL concentration of the toxins also induced alterations in the muscle fibres. It is likely that the onset of changes was triggered by damage of the phospholipid bilayer of the fibre membrane as inferred by histological and ultrastructural findings. Neuromuscular blockade and/or muscle damage, as induced by BmjeTX-I and BmjeTX-II in CBC preparations, was also reported in nerve-muscle preparations treated with other toxins, such as BthTX-I [ B. jararacussu venom and BnpTX-I and BnpTX-II toxins [ B. (neuwiedi) pauloensis venom. Muscle damage inferred by rapid elevation of plasma CK activity in mice was also observed by Ponce-Soto et al. [ in vivo with the two toxins here investigated. These results revealed that BmjeTX-I and BmjeTX-II PLA2s displayed a myotoxic effect, as the majority of venoms and toxins were isolated from Bothrops snakes, which is not associated with impairment of the nicotinic receptors of the postsynaptic sarcolemma.In this study, the 10  BthTX-I , 10 and  BthTX-I  from B. I toxins  from B. o et al.  in vivo  B. jararacussu venom. Moreover, the changes in the intramuscular motor nerve axons are additional evidence of the neurotoxic action of the two PLA2s.Also, muscle ultrastructural alterations as those induced by BmjeTX-I and BmjeTX-II are similar to those described by Rodrigues-Simioni et al.  in frog  Bothrops marajoensis venom.The absence of proteins involved in synaptic vesicles integrity (synaptophysin), vesicle docking, and transmitter exocytosis , in preparations incubated with BmjeTX-I and BmjeTX-II, is direct and strong evidence supporting our hypothesis that BmjeTX-I and BmjeTX-II affect neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal. The study reinforces the hypothesis that the lack of these proteins was responsible for the neurotoxicity caused by both toxins from