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Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes.
Author Summary
Appropriate cellular responses to external stimuli depend on the highly orchestrated activity of interconnected signaling cascades. One crucial level of control arises from the formation of discrete complexes through scaffold proteins that bind multiple components of a given pathway. Central for our understanding of these signaling platforms is the archetypical MAP kinase scaffold Ste5p, a protein that is restricted to budding yeast and close relatives. We identified HAM-5, a protein highly conserved in filamentous ascomycete fungi, as cell–cell communication-specific scaffold protein of the Neurospora crassa MAK-2 cascade (homologous to the budding yeast pheromone pathway). We also describe a network of upstream acting proteins, consisting of two Ste20-related kinases, the small G-protein RAS-2 and the adenylate cyclase capping protein CAP-1, whose signals converge on HAM-5. Our work has implications for the mechanistic understanding of MAP kinase scaffold proteins and their function during intercellular communication in eukaryotic microbes as well as higher eukaryotes.
Citation: Dettmann A, Heilig Y, Valerius O, Ludwig S, Seiler S (2014) Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5. PLoS Genet 10(11): e1004762. https://doi.org/10.1371/journal.pgen.1004762
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Received: July 3, 2014; Accepted: September 18, 2014; Published: November 20, 2014
Copyright: © 2014 Dettmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: This research project was financially supported through the DFG Heisenberg fellowship SE1054/6-1 and the DFG grant SE1054/9-1. The Fungal Genetics Stock Center (www.fgsc.net) and the Neurospora program grant PO1 GM068087 "Functional analysis of a model filamentous fungus" are acknowledged for strains. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Intercellular communication is critical for the survival of simple unicellular organisms such as bacteria and yeasts and is central for the development and function of multicellular plant and animal systems [1]–[4]. Cell-cell signaling and somatic cell fusion is also required to develop the interconnected mycelial network characteristic of filamentous fungi [5]. This feature is important for the fitness of the fungal colony by the shared use of information, nutrients and organelles between individual cells [6], [7]. Consequently, hyphal anastomosis is critical for host colonization and symbiotic interactions as well as for virulence of pathogenic species [8]–[12]. Hyphal fusion is comparable to homotypic cell fusion between genetically identical cells of higher eukaryotes, which results in the formation of multinucleate syncytia [13], [14]. Important examples for human biology are myoblast fusion during muscle differentiation, trophoblast fusion during placental development and osteoclast fusion during bone formation. Thus, fungal self-signaling may provide a powerful model for understanding molecular mechanisms of homotypic cell communication during animal and human tissue development.
In the ascomycete model mold Neurospora crassa, an unknown chemical ligand mediates chemotropic communication between genetically identical cells. Germinating spores mutually attract each other and subsequently fuse to generate an interconnected network of multinucleate cells that form the mycelial colony [5], [15]. This process of self-signaling is based on the oscillatory recruitment of the NRC-1–MEK-2–MAK-2 mitogen activated protein kinase (MAPK) cascade (homologous to the Ste11p-Ste7p-Fus3p mating pathway in budding yeast) and of SOFT, a protein of unknown molecular function, to the opposing tips of communicating germlings [16], [17]. The rapid alternation of these two different physiological states of "homing" cells likely reflects signal response and delivery, respectively. Although these findings resulted in a first qualitative model to describe the excitable behavior of the MAK-2 module [18], our understanding of oscillatory MAK-2 signaling is hampered by the fact that most components of the signaling machinery – including the postulated secreted signal and its cognate receptor(s), regulators of the MAPK cascade as well as most MAK-2 targets – are unknown.
N. crassa and other filamentous fungi possess G-protein coupled receptors, heterotrimeric G-proteins, STE20-related kinases, components of the cAMP machinery and Ras/Rho-type GTPase modules known to function upstream or in parallel of MAPK signaling in fungi and higher eukaryotes [19]–[22]. However, mutant analyses indicate that individual deletions of these components are dispensable for vegetative cell communication (summarized in [23]). We hypothesized that redundant functions between the mentioned proteins require additional approaches to classical mutant hunts in order to dissect MAK-2 signaling. In this study, we used a proteomics approach that allowed the identification of STE-50 as regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover we defined a network of proteins, consisting of two Ste20-related kinases, the small G-protein RAS-2 and the adenylate cyclase capping protein CAP-1, whose signals converge on the MAK-2 pathway.
Composition of the MAK-2 cascade
A high-quality interactome of the MAK-2 MAPK module was generated by affinity purification experiments coupled to mass spectrometry (AP-MS) with strains expressing functional GFP-fusion proteins of the three kinases of the MAK-2 cascade (Table S1). Each of the three bait proteins recovered the tripartite kinase cascade with high stringency in the two biological replicate purifications performed, indicating the suitability of the approach. Moreover, we identified two proteins, STE-50 and HAM-5, which displayed tight interactions with all three kinases (Figure 1 A).
(A) Proteins associated with the MAK-2 cascade were identified in affinity purification experiments coupled to mass spectrometry (AP-MS), and identified proteins were filtered against control purifications using GFP as bait (Table S1). Only proteins identified in two biological replicates and absent from the control data set are shown. Protein coverage by peptides and the number of identified total and unique peptides identified are given for the better of the two purifications. Bold numbers indicate the GFP-fusion protein used as bait. (B) Physical interactions between MAK-2 pathway components were mapped in yeast two-hybrid (Y2H) tests. The indicated constructs were co-expressed in strain AH109 and yeast growth was analyzed on the indicated selective media. The reciprocal Y2H assays are shown in Figure S2. (C) Summary schema of Y2H-based interactions of the indicated proteins and their domains. Color-coded lines below the protein schemas and dashed connectors indicate the used constructs and detected interactions, respectively.
The remaining hits that associated with all three kinases were only identified with poor coverage and/or in one of the two purifications and thus could constitute contaminants. However, among them were PPG-1, PP2A-A, HAM-3 and MOB-3, components of the striatin-interacting phosphatase 2A and kinase (STRIPAK) complex that is required for fungal cell-cell signaling [24]–[29]. This suggests that at least some of the additional hits may represent dynamically, and thus weakly interacting components. Thus, we assayed available mutants defective for additional candidate proteins that associated with the three kinases for tropic interaction defects and determined that the casein kinase 2 heterodimer CKA/CKB-1, the serine/threonine kinase YPK-1, type V myosin/NCU01440, and the hypothetical protein NCU06265 (designated HAM-13) were required for proper cell communication. Consistent with these defects, we also detected reduced MAK-2 phosphorylation levels in cell extracts of these five mutant strains (Table 1; Figure S1). The combined proteomics data and mutant characteristics indicate a specific involvement of these proteins in cell-cell communication despite the fact that they were identified only with poor coverage in the AP-MS analysis. However, further experiments are required to determine their mechanistic mode of action and to exclude indirect effects of the mutations on MAK-2 pathway functionality. In addition, mutants defective for the hypothetical proteins NCU00627, NCU02606, NCU02972, and NCU08957 that also interacted with the entire kinase cascade displayed inconspicuous communication patterns and were thus not tested for MAK-2 phosphorylation, because they likely represented contaminants.
Interestingly, we also determined that MAK-2, yet not the two upstream kinases interacted with multiple components of the nuclear import/export machinery (i.e. importin alpha, importin beta-1, importin beta-3, exportin-1, nuclear pore protein NCU01702; Table S1) and the two transcriptional regulators PP-1 and RCO-1, which were previously identified as key effectors of the MAK-2 pathway [30]–[32].
STE-50 is required for NRC-1 activation
Budding yeast Ste50p functions as adaptor protein of the MAP3K Ste11p that connects heterotrimeric G-proteins and small GTPases with various MAPK cascades [33], [34]. This is achieved through the modular structure of Ste50p. The protein consists of an N-terminal protein interaction domain called the sterile alpha motif (SAM) [35], [36] and a C-terminal Ras association (RA) domain that can bind to small Ras and Rho-type GTPases and is required for membrane delivery of the Ste11p/Ste50p complex [37], [38]. Homologs in filamentous fungi are not as well characterized, but have been proposed to function as scaffold proteins in MAPK cascades [39]–[42]. Yeast two hybrid (Y2H) assays confirmed the physical interaction of STE-50 with itself and with NRC-1, yet not MEK-2 and MAK-2, through the SAM domains present in the two proteins (Figure 1 B, C; Figure S2). The presence of a stable STE-50/NRC-1 complex was further supported by the reciprocal AP-MS analysis of GFP-STE-50 interacting proteins, which revealed a stable interaction of STE-50 with NRC-1, but not other components of the MAPK cascade (Figure 1 A).
Strains expressing gfp-ste-50 under its native promoter complemented the defects of the deletion strain, but barely showed any fluorescence, which was consistent with the weak expression profile of its binding partner NRC-1 [17]. Thus, we analyzed strains expressing the fusion construct under the control of the Pccg-1 promoter. GFP-STE-50 displayed a subcellular dynamics similar to that previously described for the three kinases of the MAK-2 cascade [16], [17] and accumulated at the future fusion site after the two germlings had established physical contact (Figure 2 A). Consistent with the expression profile and localization pattern described for NRC-1 [17], we observed only weak cytoplasmic localization of GFP-STE-50 in mature hyphae and exclusion of the fusion protein from nuclei. However, GFP-STE-50 accumulated at septa and contact sites of fusing hyphae within an established colony (Figure 2 B).
(A) GFP-STE-50 localizes in a dynamic manner to opposing tips of two communicating germlings and the site of cell-cell contact. The oscillation period is approximately three to five minutes. See Video S1 for time course. (B) GFP-STE-50 localizes in a diffuse cytosolic manner in non-communicating hyphae and is excluded from nuclei (left image). Moreover, GFP-STE-50 accumulates at septa (arrowheads) and the contact point (arrow) of communicating hyphal tips (right image). (C) Δste-50 fully phenocopies Δmak-2 defects (see Figure S3 for comparative characterization of mutant defects), and the mutant characteristics are complemented by expression of a constitutive active NRC-1(P488S) allele in Δste-50. The slant images represent macroscopic appearance and conidiation pattern, while the Table summarizes rates of mycelial extension, protoperithecia (PP) formation, germling communication and MAK-2 activities of the indicated strains.
We determined that a Δste-50 deletion mutant fully phenocopied all defects described for defective MAK-2 signaling [31], [43], [44] and lacked detectable MAK-2 kinase activity (Table 1; Figure 2 C; Figure S3). Based on these characteristics of the mutant and the tight interaction of STE-50 with NRC-1, we hypothesized that STE-50 may function as regulatory subunit of the MAP3K. To test this, we expressed a recently generated [17], constitutive active NRC-1 version NRC-1(P488S) in Δste-50. This construct fully complemented all defects of the deletion strain and confirmed our hypothesis (Figure 2 B).
HAM-5 functions as cell communication-specific scaffold of the MAK-2 module
Ham-5 was originally identified in a genetic screen for N. crassa mutants that fail to undergo cell fusion [30]. Its closest homolog, Podospora anserina IDC1, is required for NADPH-oxidase-dependent nuclear accumulation of the cell wall integrity MAPK Mpk1 [45], but the mechanistic basis underlying this observation remains unresolved. MAK-2 activity was reduced to ca. 1/10 of wild type in the Δham-5 mutant (Table 1; Figure S3), indicating significantly compromised, but not abolished MAK-2 pathway function in this strain. We observed increased mycelial extension rates of Δham-5 when compared to Δste-50 or Δmak-2, residual tropic interactions (≤10% of wild type) and the retained capacity to produce low amounts of infertile protoperithecia (ca. 20% of wild type; Figure S3). Co-immunoprecipitation experiments were used to confirm the interaction of HAM-5 with all three kinases of the MAK-2 pathway Figure S2). In order to further dissect the physical interaction pattern of these proteins, we performed Y2H tests. In these assays, we determined that HAM-5, which contained seven N-terminally located WD40 repeats and two short coiled coil regions in the C-terminal region, was able to homo-dimerize and also interacted with STE-50 and with MAK-2, yet not with MEK-2 and NRC-1 (Figure 1 B, C; Figure S2).
Functional Pccg-1-driven HAM-5-GFP displayed the predicted dynamic subcellular localization during germling communication and strictly co-localized with MAK-2 as dynamically forming intracellular complexes that associated with the communicating tips of the two cells with an oscillation period of three to five minutes (Figure 3 A, B). At least some of the tip-associated signal was generated by recruitment of cytosolic HAM-5- and MAK-2-containing puncta to the apex (Figure 3 C). Diffuse HAM-5-GFP label was excluded from nuclei, and bright HAM-5 puncta were not obviously associated with nuclei. However, we detected weakly labeled HAM-5 puncta that decorated nuclear envelopes (preferentially marking those nuclei that localized closely to the cell tip) simultaneously with the first appearance of tip-associated HAM-5 signal. (Figure 3 D).
(A) Motile HAM-5-GFP puncta appear in a dynamic manner at opposing tips and in the cell body of two communicating germlings with an oscillation period of approximately three to five minutes and subsequently accumulate at the contact point of the two tips. See Video S2 for time course. Note that the diffuse cytoplasmic label of HAM-5-GFP decreased during the formation of HAM-5 puncta. Arrowheads indicate nuclei, from which HAM-5 is excluded. (B) HAM-5 and MAK-2 co-localize to puncta at the cell tip and within the germling (marked by arrows). MAK-2, but not HAM-5 also accumulates in nuclei (marked by arrowheads). Note that mCHERRY-tagged HAM-5 is strongly targeted to vacuoles for degradation (marked by red arrows). See Video S3 for time course. (C) Movement of a HAM-5/MAK-2 double-labeled spot towards the cell tip. See Video S3 for time course. (D) HAM-5 puncta can associate with nuclear envelopes (enlarged insert). See Video S4 for time course. (C) Movement of a HAM-5/MAK-2 double-labeled spot towards the cell tip. See Video S3 for time course.
Dynamic co-recruitment of HAM-5 and MAK-2 to communicating hyphal tips, septa and intracellular puncta was also observed in the mature colony (Figure 4 A, Figure S4). Moreover, HAM-5 and SOFT oscillated with opposing recruitment phases in communicating hyphae (Figure S4), indicating that the molecular machinery required for germling communication is also operating during hyphal anastomosis within the mature colony. In contrast to MAK-2 [17], HAM-5 was not detected at the apex of non-communicating hyphae (Figure 4 B). We therefore asked if MAK-2 activity is required for HAM-5 aggregation during cell signaling. When we localized HAM-5-GFP in Δmak-2 germlings, we observed the presence of HAM-5-GFP puncta, which remained stable over long time periods (Figure 4 C). Moreover, we noticed that intracolonial Δmak-2 hyphae that had (by chance) established physical contact grew in parallel over longer distances with HAM-5-GFP puncta formed primarily in one of the two hyphae, while septal pores were strongly labeled in the second cell (Figure 4 C). Quantification of this phenomenon revealed that 72±7% (n = 141) of the analyzed intracolonial hyphal pairs displayed this HAM-5 distribution. Thus, MAK-2 is essential to regulate the subcellular dynamics of HAM-5, but complex formation does not require the MAPK.
(A) Dynamic HAM-5-GFP oscillation is observed in approaching tips within the established mycelium. Dynamic localization of HAM-5 to septa is indicated by arrows, and septum-associated HAM-5-GFP puncta are enlarged in the first image (insert co-labeled with FM4-64 to show plasma membrane and septum). See Video S5 for time course. (B) HAM-5 is not enriched at hyphal tips of non-communicating hyphae, although HAM-5-GFP spots are visible throughout the cytoplasm of the growing hypha. Plasma membrane and Spitzenkörper are labeled with FM4-64. (C) HAM-5-GFP puncta are also formed in Δmak-2 hyphae (left panel) and germlings (right panel), although the protein does not concentrate at germling tips. Note the distinct localization patterns of HAM-5-GFP in the two contacting hyphae in a Δmak-2 colony: multiple HAM-5 puncta are visible the approaching (left) hypha, while primarily the septal pores are labeled in the contacted (right) hypha and only few puncta are visible. (D) Reciprocal co-immunoprecipitation experiments of the tagged kinases flag-NRC-1 and MAK-2-GFP indicate interaction of the MAPK module in wild type but not in the Δham-5 mutant.
We reasoned that the interaction pattern of HAM-5 with multiple MAK-2 pathway components and its localization dynamics were consistent with a scaffold function of HAM-5 for the MAK-2 cascade. We tested this hypothesis by performing co-immunoprecipitation experiments (Figure 4 D). NRC-1 and MAK-2 co-precipitated when tagged versions of both proteins were co-expressed in wild type, while we were unable to detect interactions between the two kinases in a Δham-5 background, indicating that HAM-5 is critical for maintaining the integrity of the kinase cascade.
A network of Ste20-related kinases, RAS-2 and the adenylate cyclase capping protein CAP-1 signals upstream of the MAK-2 pathway
Our AP-MS analysis identified several proteins that specifically interacted with NRC-1, yet not MEK-2 or MAK-2 and thus could constitute upstream components of the MAK-2 pathway. Among them were the MAP4K STE-20, the small GTPase RAS-2/SMCO-7 and the capping protein CAP-1/NCU08008 of the adenylate cyclase (AC) complex (Table S1). A fully functional STE-20-GFP fusion construct (Figure S5) recovered STE-50, NRC-1, HAM-5 and RAS-2 in reciprocal AP-MS experiments (Figure 1 A), and Y2H assays confirmed physical interactions of STE-20 with HAM-5, NRC-1 and STE-50 (Figure 1 B, C; Figure S2). Moreover, NRC-1 and weakly also STE-50 interacted with RAS-2 through the Ras-association domains present in both proteins in Y2H tests. We did not detect any Y2H interaction of STE-50 and NRC-1 with the second Ras-type GTPase, RAS-1/NCU08823 (also called BAND; [46]), present in N. crassa, underscoring the specificity of the MS and Y2H analyses. We also detected MST-1, a recently identified accessory Ste20-related kinase of the septation initiation network, which localized to spindle pole bodies and septa [47], [48] as NRC-1- and MEK-2-interacting protein by AP-MS and Y2H analysis (Table S1; Figure 1 B). However, communication frequencies and MAK-2 activities of Δmst-1 and of a generated Δste-20;Δmst-1 double mutant indicated that MST-1 is not critically required for MAK-2 signaling (Table 1).
Consistent with an involvement in cell-cell communication, we found that STE-20-GFP formed membrane-associated apical crescents in mature hyphae, was enriched in a stable manner at both communicating germling tips and localized at the contact point of interacting cells in addition to its association with septa in germlings as well as mature hyphae (Figure 5 A; Figure S5). Moreover, a functional GFP-RAS-2 construct distributed along the entire plasma membrane in germlings and hyphae and localized to the contact point of interacting germlings (Figure 5 B; Figure S5). As predicted for components that signal upstream of the MAK-2 cascade, we observed reduced MAK-2 activities and cell communication frequencies in Δste-20, in Δras-2, the previously described ras-2 allele smco-7 [49], and in two mutants affecting the predicted RAS-activating GDP-GTP-exchange factor CDC-25 (Table 1; Figure 5 C). Only residual levels of germling communication and MAK-2 activity were detected in a Δste-20;Δras-2 double mutant, highlighting the joint importance of RAS-2 and STE-20 for self-signaling. The MAK-2 pathway is also induced by other external stimuli, such as reactive oxygen species [17], [28], and we asked if the identified proteins were also required for stress-induced activation of MAK-2. We determined that H2O2-induced activation of the MAK-2 pathway was abolished in Δras-2 and in the Δste-20;Δras-2 double mutant (Figure 5 D), indicating that both proteins are general components of the MAK-2 pathway.
(A) STE-20-GFP localizes in a stable manner to the apices of two communicating germlings and marks the site of contact. See Video S6 for time course. (B) GFP-RAS-2 associates with the entire plasma membrane of germinating spores and localizes at the contact point of two communicating cells. See Video S7 for time course. (C) Time course experiments to determine rates of germination and of chemotropic interactions of the indicated mutants. Note that due to delayed germination Δcr-1 and Δcap-1 strains are assayed at later time points than the other mutants. (D) Quantification of basal and stress-induced MAK-2 phosphorylation levels (detected with p42/44 antibodies) in cell extracts of exponentially growing liquid cultures of the indicated strains (n = 3). A representative Western blot is depicted below; tubulin was used as loading control.
We also tested for binding of these components to the AC capping protein CAP-1 and detected a positive Y2H interaction with STE-50, but not with any of the other proteins of the MAK-2 pathway (Figure 1 B). This may indicate that our AP-MS-based identification of CAP-1 in precipitates of STE-50, NRC-1 and STE-20 (Table S1; Figure 1 A) was mediated through STE-50. Nevertheless, we observed altered cell communication frequency and reduced MAK-2 activity in Δcap-1, consistent with a functional involvement of CAP-1 in MAK-2 signaling (Table 1). Significantly, we also detected reduced tropic germling interactions in the AC mutant Δcr-1 (Figure 5 C), indicating that cAMP signaling is involved in, but is not essential for cell-cell communication. These data are consistent with a previous report, which showed that cr-1 germlings are able to generate conidial anastomosis tubes, although no quantitative analysis was performed in this study [50].
Despite considerable progress in recent years, our mechanistic understanding of oscillatory MAK-2 behavior during homotypic cell communication is hampered by the fact that many components of the signal transduction machinery are still unknown and that the molecular functions of proteins known to be required for signaling are only poorly understood. One important finding of this study is the identification of STE-50 and HAM-5 as central components of the MAK-2 pathway (Figure 6). We propose STE-50 as tightly associated, regulatory subunit of NRC-1 and HAM-5 as scaffold protein of the MAK-2 cascade. Based on our data, STE-50 may have both NRC-1-activating as well as targeting functions, and we currently cannot rule out any of these hypotheses. Nevertheless, RAS-2 interacts with both NRC-1 and STE-50 in Y2H assays, and thus STE-50 may not be essential for membrane targeting, yet full complementation of Δste-50 by expression of constitutive active NRC-1(P488S) indicates that STE-50 is critical for activation of the MAP3K. We do not have any evidence for a scaffold function of STE-50 in N. crassa, contrasting data obtained in other filamentous fungi, which have suggested interactions of STE-50 homologs with other kinases in addition to the MAP3K [40], [42], [51].
Ligand-induced activation of an unknown receptor may be transmitted through plasma membrane-associated STE-20, RAS-2 and CAP-1, which signal toward the NRC-1/STE-50 complex and recruit the MAPK cascade through activation and clustering of the scaffold HAM-5 at intracellular puncta. MAK-2 activation triggers nuclear gene expression through interaction with the transcription factor PP-1 and the RCO-1/RCM-1 complex and the cytosolic activation of the secretory pathway and cell polarity machineries to coordinate pulsed signal release and chemotrophic growth towards the partner cell, respectively. MAK-2 activity is also required for termination of the receiver phase, potentially through negative feedback phosphorylation of the MAPK and disassembly of the MAK-2/HAM-5 module. MAK-2 pathway function may also be regulated through the STRIPAK complex, the CK2 heterodimer, membrane lipid composition, the septum-associated septation initiation network SIN and motor protein-dependent vesicle trafficking.
Central for our understanding of scaffold proteins is the archetypical MAP kinase scaffold Ste5p of the yeast mating pathway [52], [53]. However, Ste5p is restricted to budding yeast and close relatives [20], [54]. In contrast, homologs of HAM-5 are detected in all sequenced members of the Pezizomycotina subphylum (the group of filament-forming ascomycete fungi), but are absent from the genomes of unicellular ascomycete fungi (Figure S6). Thus, we propose HAM-5 as scaffold of the N. crassa MAK-2 cascade and of homologous MAPK modules in other filamentous fungi. This hypothesis is further strengthened by an accompanying study, which also identified HAM-5 as scaffold protein of the MAK-2 pathway [55]. We hypothesize that HAM-5 and MAK-2 are co-recruited to intracellular puncta in the presumed signal receiver phase [15], [16] and that some these puncta are subsequently targeted to the apical region of communicating cells. This process may potentially reflect the predicted auto-amplification of the incoming signal through the MAK-2 cascade or priming of the receiving cell for signal release during the next phase of communication [18]. The formation of HAM-5 puncta in the Δmak-2 mutant indicates that MAK-2 is not required for complex formation of HAM-5. HAM-5 puncta remain stable over long time periods in Δmak-2, and hyphae in contact frequently display distinct HAM-5 localization patterns, suggesting that the two cells are locked into two distinct signaling modes. Thus, complex dispersion and the switch from signal receiving to signal sending require MAK-2 activity. One possible mechanism for complex disassembly and termination of the signal receiving phase of the cell may involve accumulating phosphorylation of the HAM-5 scaffold through MAK-2 and/or additional unknown kinases as proposed in [55]. This hypothesis is also in line with previous work reporting that unregulated (both reduced as well as increased) MAK-2 activity allows reasonable tip growth of vegetative hyphae, while cell-cell communication requires the regulated on/off switch of the MAK-2 cascade [17], [30]. In contrast to MAK-2 [17], HAM-5 did not accumulate at the apex of non-communicating hyphae, and residual MAK-2 pathway functionality is retained in Δham-5 allowing reasonable rates of mycelial extension. These data imply a cell communication-specific function of HAM-5 for the MAK-2 cascade. In addition, previous work had identified the kinase adapter HYM-1/MO25, which associates with multiple Ste20-related kinases in N. crassa (this study; [17], [47]) and higher eukaryotes [56] as general platform that is required for MAK-2-dependent intercellular communication and for basic growth-associated functions of the MAK-2 pathway.
We identified STE-20 and RAS-2 that together are critical for signal input of the MAK-2 pathway. Moreover, the AC capping protein CAP-1 is also involved in cell-cell communication. The yeast homolog of CAP-1, Srv2p, was identified as part of a RAS-responsive AC complex in S. cerevisiae [57], [58]. CAP-1 homologs also play a critical role in regulating actin dynamics and cell polarity in various fungi as well as higher eukaryotes [59]–[62]. Thus, the association of STE-50 with CAP-1 may link upstream components of the MAK-2 pathway with Ras/cAMP signaling and with cell morphogenesis through regulation of actin dynamics. We did not detect the second STE-20-related kinase CLA-4 as MAK-2 pathway-associated protein in our proteomics analysis, consistent with previous Y2H data that indicate no physical interaction between NRC-1 and CLA-4 [17]. Thus, CLA-4, which was recently implicated in self-signaling in N. crassa [63], may function as part of another module, such as the predicted BEM-1/CDC-42/RAC/CDC-24/CLA-4 complex that regulates cell polarity and potentially also chemotrophic growth [64]-[66]. Intriguingly, we also identified the Ste20-related kinase MST-1 as NRC-1- and MEK-2-interacting protein (this study; [47]). Although the significance of these interactions is currently unclear, the entire MAK-2 cascade including STE-50 and HAM-5 associates with septa ([16], [17]; this study). The dynamic localization of HAM-5 at septa during intra-colony communication and its strong septal pore association may therefore indicate that MAK-2-dependent signals can originate from septa and/or that incoming signals are integrated by the MAPK pathway at these sites. This speculation is supported by accumulating evidence of central functions of septal pores as signaling hubs within the fungal colony [67]–[69].
Finally, the association of the casein kinase 2 holoenzyme and the PP2A heterotrimer with the MAK-2 cascade opens the intriguing possibility for activity regulation of the MAPK cascade. Analogous results were obtained for the mammalian casein kinase 2, which also associates with phosphatase 2A and down-regulates the PP2A substrate MEK1 [70], [71]. A similar, regulatory role may also be attributed to the recently defined STRIPAK complex [27], [28], [72]. Significantly, we did not detect the STRIPAK subunits HAM-2 and HAM-4 in our MS analysis, and thus only a sub-complex consisting of the PP2A heterotrimer and the kinase adaptor protein MOB-3 may associate with and regulate the MAK-2 cascade. MAK-2 pathway regulation may also occur through modulation of lipid composition and thus membrane dynamics, which is central for the organization and dynamic localization of multiple signal transduction pathways, including the yeast pheromone pathway [73], and has also been implicated in self-signaling in N. crassa [74]. Yeast homologs of the N. crassa kinases YPK-1 and NRC-2 function as regulators of so-called flippase complexes [75], [76], which are primarily localized to the plasma membrane at sites of polarized growth, and phospholipid flipping has been shown to regulate Cdc42p signaling during polarized growth in yeast [77]. Because N. crassa ypk-1 and nrc-2 strongly phenocopy vegetative and developmental traits of MAK-2 pathway mutants [78], [79], the association of YPK-1 with NRC-1 and the cell communication defects detected in deletion and temperature-sensitive ypk-1 mutants will be of particular interest for dissecting oscillatory MAK-2 signaling and the chemotropic behavior of communicating cells.
Materials and Methods
Strains, media and tagged protein constructs
Strains and oligonucleotides used in this study are listed in Tables S2 and S3, respectively. General genetic procedures and media used in the handling of N. crassa are available through the Fungal Genetic Stock Center (www.fgsc.net). Fusion proteins were ectopically expressed under the control of the ccg-1 or gpd-1 promoters at the his-3 locus. The ORFs of ste-50, ham-5, ste-20 and ras-2 were amplified by PCR as annotated by the N. crassa database and introduced into pMF272, pNGFP and pJV15-2 [80]–[82]. For co-expression of tagged proteins, the pccg-1-sgfp sequence of the vector pNGFP [81] was replaced with the inversely oriented ccg-1 and gpd-1 promoters amplified from the vector pBiFC [83] to generate the vector pCCG1-pGPD1, which allowed insertion of fusion-constructs via SgsI/SpeI (mak-2-sgfp) and SwaI/EcoRI (3xflag-nrc-1) amplified from the template plasmids pMF272-mak2 and pFLAG-nrc1 [17]. Resulting plasmids were transformed into his-3 and/or his-3;Δ strains. Complementation of tropic interaction, growth and developmental defects of the deletion strain was used to confirm functionality of the constructs.
Protein methods
Co-immunoprecipitation experiments were performed as described [28], [84]. Conditions and plasmids used for the Y2H assays are specified in [83], [85]. Basal and stress-induced MAK-2 activity of exponentially growing N. crassa liquid cultures was determined using polyclonal rabbit α-Phospho-p44/42 MAPK (Cell Signaling Technology, USA) and goat α-rabbit IgG-HRP (Santa Cruz, USA) as primary and secondary antibodies, respectively as described [86], [87]. Briefly, exponentially growing, liquid cultures were harvested gently by filtration using a Büchner funnel and ground in liquid nitrogen. The frozen mycelial powder was incubated in 95% ethanol at -20°C for ≥12 h, the supernatant removed after centrifugation and the pellet vacuum-dried in a SpeedVac concentrator (Thermo Fisher Scientific, USA). extraction buffer (50 mM Tris/HCL pH 7,5, 100 mM KCl, 10 mM MgCl2, 0.15% NP-40, 5 mM NaF, 1 mM PEFA, 1 mM Na3VO4, 25 mM β-glycerophosphate, 2 mM benzamidine, 2 ng/µl pepstatin A, 10 ng/µl aprotinin, 10 ng/µl leupeptin) was added, the samples mixed and incubated at 80°C for 5 min and the supernatant collected after centrifugation. After a second round of extraction, the supernatants pooled, subjected to another centrifugation step, and the protein concentration determined using a Nanodrop spectrophotometer (ND-1000, Peqlab, Germany). Sample volumes corresponding to 75 µg total protein per lane were subjected to SDS polyacrylamide gel electrophoresis. ≥3 biological replicates were quantified for each experiment. For quantification of MAK-2 phosphorylation levels, exposed films were scanned at a resolution of 600 dpi and densitometry was performed on the resulting tif files employing the AIDA Image Analyzer (version 4.22; raytest Isotopenmessgeräte, Germany) in transmission mode. Intensity values [arbitrary units] measured within a region of interest of fixed size containing the MAK-2 protein bands were corrected by subtraction of local background, normalized to the protein amount loaded and used for further evaluation.
GFP-trap experiments, mass spectrometry and database analysis were performed as described [28], [88]. Pulverized mycelium was mixed 1∶1 with extraction buffer and centrifuged (1 h, 10,000 rpm, Sorvall SS34 rotor) in order to obtain crude cell extracts. Cell extracts were incubated with 2 µl GFP-trap beads (Chromotek, Germany) per 15 ml cell extract on a rotator for 2 h at 4°C. The beads were washed three times with IP buffer, associated proteins were recovered by boiling them in Laemmli buffer and separated by SDS polyacrylamide gel electrophoresis analysis. Peptides of in-gel trypsinated proteins were extracted from Coomassie-stained gel slices. Peptides of 5 µl sample solution were trapped and washed with 0.05% trifluoroacetic acid on an Acclaim PepMap 100 column (75 µm×2 cm, C18, 3 µm, 100 Å, P/N164535 Thermo Scientific, USA) at a flow rate of 4 µl/min for 12 min. Analytical peptide separation by reverse phase chromatography was performed on an Acclaim PepMap RSLC column (75 µm×15 cm, C18, 3 µm, 100 Å, P/N164534 Thermo Scientific, USA) running a gradient from 96% solvent A (0.1% formic acid) and 4% solvent B (acetonitrile, 0.1% formic acid) to 50% solvent B within 25 min at a flow rate of 250 nl/min. Peptides eluting from the chromatographic column were on-line ionized by nano-electrospray using the Nanospray Flex Ion Source (Thermo Scientific, USA) and transferred into the mass spectrometer. Full scans within m/z 300–1850 were recorded by the Orbitrap-FT analyzer at a resolution of 60,000 at m/z 400. Each sample was analyzed using two different fragmentation techniques applying a data-dependent top 5 experiment: collision-induced decay with multistage activation and readout in the LTQ Velos Pro linear ion trap, and higher energy collision dissociation and subsequent readout in the Orbitrap-FT analyzer. LC/MS method programming and data acquisition was performed with XCalibur 2.2 (Thermo Scientific, USA). Orbitrap raw files were analyzed with Proteome Discoverer 1.3 (Thermo Scientific, USA) using the Mascot and Sequest search engines against the N. crasssa protein database with the following criteria: peptide mass tolerance 10 ppm, MS/MS ion mass tolerance 0.8 Da, and up to two missed cleavages allowed.
Spinning disc confocal microscopy was performed as described [47] using an inverted Axio Observer Z1 microscope (Zeiss, Germany) equipped with a CSU-22 confocal scanner unit and a CCD camera (Axiocam MRm Rev.3). ZEN Blue 2012 software (Zeiss, Germany) was used for image/video acquisition and image analysis. Plasma membrane was stained with FM4-64 (1 mg/ml-1), and time-lapse imaging was performed at capture intervals of 20 s for periods up to 40 min using a C-Apochromat 63x/1.2 W objective. Image series were converted into movies (*.mov).
Tropic germling interactions of ≥100 germlings in each of 2-3 biological replicates per experiment were quantified as described [28], [65]. In brief, strains were grown on Vogel's minimal media slants for 7 days at 26°C. Conidia were harvested with 1 to 2 ml H2O, and the conidial suspension was filtered through cheesecloth. A total number of 5×106 fresh spores were spread out on a minimal media plate, incubated at 30°C and analyzed after the indicated time points using a Zeiss Axiophot 2 microscope with a Zeiss Plan-Apochromat 63x/1.40 oil immersion objective. Only germlings that had produced germ-tubes of at least 2 µm length and were localized within a distance smaller than 10 µm to another germling were included in the trophic interaction analysis.
Supporting Information
Figure S1.
Mutant characteristics of MAK-2 pathway components. (A) Pictures of communicating germlings of the indicated strains were taken after 4-6 h germination at 30°C. (B) MAK-2 phosphorylation levels were determined with p42/44 antibodies in cell extracts of exponentially growing liquid cultures of the indicated strains. Tubulin was used as loading control.
Figure S2.
Interaction network of the MAK-2 pathway. (A) Physical interactions between MAK-2 pathway components were mapped in yeast two-hybrid (Y2H) tests. The indicated constructs were co-expressed in strain AH109 and yeast growth was analyzed on the indicated selective media. (B) Reciprocal co-immunoprecipitation experiments from cell extracts co-expressing the functionally tagged proteins NRC-1, MEK-2 and MAK-2 and HAM-5 indicate interaction of all three kinases with HAM-5.
Figure S3.
Comparative characterization of Δste-50 and Δham-5. Analysis of macroscopic appearance (A), mycelial extension rate (B), sexual development (C), germling communication frequency (D), and basal/stress-stimulated MAK-2 activity level (E) indicates that Δste-50 fully phenocopies Δmak-2 defects, while Δham-5 retains residual MAK-2 pathway functionality.
Figure S4.
Co-localization of HAM-5 with the SOFT/MAK-2 cell communication machinery in communicating hyphae within the established mycelium. (A) MAK-2-GFP and HAM-5-mCherry co-localize in a dynamic manner to opposing tips of two communicating hyphae. Note the dynamic nuclear accumulation of MAK-2, yet not HAM-5 in the presumed signal receiver phase. (B) Dynamic recruitment to communicating hyphal tips is also observed for SOFT. (C) HAM-5-mCherry and SO-1-GFP oscillate with strictly opposing recruitment phases in communicating hyphae and do not co-localize.
Figure S5.
Characterization of ste-20 (left) and ras-2 (right panels). (A) Macroscopic appearance and mycelial extension rates of the indicated deletion mutants and complemented strains. (B) Localization of GFP fusion constructs in mature hyphae. See Videos S8 and S9 for time courses. Plasma membrane and Spitzenkörper are labeled with FM4-64. H1-RFP was used to label nuclei (left panel only).
Figure S6.
Phylogram of fungal HAM-5 homologs. The tree was generated by using ClustalX 2.1 with bootstrap support (111 random number generator seed and 1000 bootstrap trials) and predicted protein sequences from selected ascomycete proteins. Note that only the WD40 domains of each protein were included in this analysis in order to use the WD40 domain of N. crassa HAM-3 as outgroup member (similar tree topologies were obtained with full length sequences; multiple alignment parameters: gap opening 10.0, gap extension 0,2, delay divergent sequences 30%, protein weight matrix gonnet series).
Table S1.
AP-MS analysis of the MAK-2 pathway.
Table S2.
N. crassa strains used in this study.
Table S3.
Oligonucleotides used in this study.
Video S1.
Time-course of GFP-STE-50 localization during germling communication and chemotrophic growth. GFP-STE-50 is recruited in an oscillatory manner to the tips of communicating germlings.
Video S2.
Time-course of HAM-5-GFP localization during germling communication and chemotrophic growth. HAM-5-GFP is recruited in an oscillatory manner to the tips of communicating germlings.
Video S3.
Time-course of HAM-5-mCHERRY and MAK-2-GFP co-localization during germling communication and chemotrophic growth. Both proteins are co-recruited in an oscillatory manner to the tips of communicating germlings.
Video S4.
Time-course of HAM-5 puncta formation at the nuclear envelope during germling communication. HAM-5 and histone H1 are tagged with GFP and RFP, respectively.
Video S5.
Time-course of HAM-5-GFP localization in the established colony. HAM-5-GFP is recruited in an oscillatory manner to the tips and septa of communicating hyphae.
Video S6.
Time-course of STE-20-GFP localization during germling communication and chemotrophic growth. STE-20-GFP is enriched at both approaching tips and accumulates at the contact point of the two cells.
Video S7.
Time-course of GFP-RAS-2 localization during germling communication and chemotrophic growth. GFP-RAS-2 associates with the plasma membrane and strongly accumulates at the contact point of the two cells.
Video S8.
Time-course of STE-20-GFP localization at the tip of mature hyphae. STE-20-GFP form a dynamic apical crescent and subapical collar at the hyphal tip.
Video S9.
Time-course of GFP-RAS-2 localization at the tip of mature hyphae. GFP-RAS-2 associates with the entire plasma membrane in a non-specific manner in established hyphae.
We thank Klaus Palme and the Institute of Biology II for providing lab space for the group.
Author Contributions
Conceived and designed the experiments: AD YH SS. Performed the experiments: AD YH OV SL. Analyzed the data: AD YH SS. Contributed reagents/materials/analysis tools: AD YH OV SL. Wrote the paper: AD YH SS.
- 1. Van Norman JM, Breakfield NW, Benfey PN (2011) Intercellular communication during plant development. Plant Cell 23: 855–864.
- 2. Celiker H, Gore J (2013) Cellular cooperation: insights from microbes. Trends Cell Biol 23: 9–15.
- 3. Kornberg TB, Roy S (2014) Communicating by touch - neurons are not alone. Trends Cell Biol 24: 370–376.
- 4. Bloemendal S, Kuck U (2013) Cell-to-cell communication in plants, animals, and fungi: a comparative review. Naturwissenschaften 100: 3–19.
- 5. Leeder AC, Palma-Guerrero J, Glass NL (2011) The social network: deciphering fungal language. Nat Rev Microbiol 9: 440–451.
- 6. Simonin A, Palma-Guerrero J, Fricker M, Glass NL (2012) Physiological significance of network organization in fungi. Eukaryot Cell 11: 1345–1352.
- 7. Richard F, Glass NL, Pringle A (2012) Cooperation among germinating spores facilitates the growth of the fungus, Neurospora crassa. Biol Lett 8: 419–422.
- 8. Craven KD, Velez H, Cho Y, Lawrence CB, Mitchell TK (2008) Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola. Eukaryot Cell 7: 675–683.
- 9. Charlton ND, Shoji JY, Ghimire SR, Nakashima J, Craven KD (2012) Deletion of the fungal gene soft disrupts mutualistic symbiosis between the grass endophyte Epichloe festucae and the host plant. Eukaryot Cell 11: 1463–1471.
- 10. Roca MG, Weichert M, Siegmund U, Tudzynski P, Fleissner A (2012) Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity. Fungal Biol 116: 379–387.
- 11. Eaton CJ, Cox MP, Scott B (2011) What triggers grass endophytes to switch from mutualism to pathogenism? Plant Sci 180: 190–195.
- 12. Ishikawa FH, Souza EA, Shoji JY, Connolly L, Freitag M, et al. (2012) Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen. PLoS One 7: e31175.
- 13. Oren-Suissa M, Podbilewicz B (2010) Evolution of programmed cell fusion: common mechanisms and distinct functions. Dev Dyn 239: 1515–1528.
- 14. Aguilar PS, Baylies MK, Fleissner A, Helming L, Inoue N, et al. (2013) Genetic basis of cell-cell fusion mechanisms. Trends Genet 29: 427–437.
- 15. Read ND, Lichius A, Shoji JY, Goryachev AB (2009) Self-signalling and self-fusion in filamentous fungi. Curr Opin Microbiol 12: 608–615.
- 16. Fleissner A, Leeder AC, Roca MG, Read ND, Glass NL (2009) Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion. Proc Natl Acad Sci U S A 106: 19387–19392.
- 17. Dettmann A, Illgen J, Maerz S, Schurg T, Fleissner A, et al. (2012) The NDR kinase scaffold HYM1/MO25 is essential for MAK2 map kinase signaling in Neurospora crassa. PLoS Genet 8: e1002950.
- 18. Goryachev AB, Lichius A, Wright GD, Read ND (2012) Excitable behavior can explain the "ping-pong" mode of communication between cells using the same chemoattractant. Bioessays 34: 259–266.
- 19. Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, et al. (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68: 1–108.
- 20. Rispail N, Soanes DM, Ant C, Czajkowski R, Grunler A, et al. (2009) Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi. Fungal Genet Biol 46: 287–298.
- 21. Qi M, Elion EA (2005) MAP kinase pathways. J Cell Sci 118: 3569–3572.
- 22. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, et al. (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422: 859–868.
- 23. Read ND, Goryachev AB, Lichius A (2012) The mechanistic basis of self-fusion between conidial anastomosis tubes during fungal colony initiation. Fungal Biology Reviews 26: 1–11.
- 24. Xiang Q, Rasmussen C, Glass NL (2002) The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa. Genetics 160: 169–180.
- 25. Maerz S, Dettmann A, Ziv C, Liu Y, Valerius O, et al. (2009) Two NDR kinase-MOB complexes function as distinct modules during septum formation and tip extension in Neurospora crassa. Mol Microbiol 74: 707–723.
- 26. Simonin AR, Rasmussen CG, Yang M, Glass NL (2010) Genes encoding a striatin-like protein (ham-3) and a forkhead associated protein (ham-4) are required for hyphal fusion in Neurospora crassa. Fungal Genet Biol 47: 855–868.
- 27. Bloemendal S, Bernhards Y, Bartho K, Dettmann A, Voigt O, et al. (2012) A homologue of the human STRIPAK complex controls sexual development in fungi. Mol Microbiol 84: 310–323.
- 28. Dettmann A, Heilig Y, Ludwig S, Schmitt K, Illgen J, et al. (2013) HAM-2 and HAM-3 are central for the assembly of the Neurospora STRIPAK complex at the nuclear envelope and regulate nuclear accumulation of the MAP kinase MAK-1 in a MAK-2-dependent manner. Mol Microbiol 90: 796–812.
- 29. Fu C, Iyer P, Herkal A, Abdullah J, Stout A, et al. (2011) Identification and characterization of genes required for cell-to-cell fusion in Neurospora crassa. Eukaryot Cell 10: 1100–1109.
- 30. Aldabbous MS, Roca MG, Stout A, Huang IC, Read ND, et al. (2010) The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa. Microbiology 156: 2621–2629.
- 31. Li D, Bobrowicz P, Wilkinson HH, Ebbole DJ (2005) A mitogen-activated protein kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa. Genetics 170: 1091–1104.
- 32. Leeder AC, Jonkers W, Li J, Glass NL (2013) Early colony establishment in Neurospora crassa requires a MAP kinase regulatory network. Genetics 195: 883–898.
- 33. Chen RE, Thorner J (2007) Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1773: 1311–1340.
- 34. Saito H (2010) Regulation of cross-talk in yeast MAPK signaling pathways. Curr Opin Microbiol 13: 677–683.
- 35. Qiao F, Bowie JU (2005) The many faces of SAM. Sci STKE 2005: re7.
- 36. Grimshaw SJ, Mott HR, Stott KM, Nielsen PR, Evetts KA, et al. (2004) Structure of the sterile alpha motif (SAM) domain of the Saccharomyces cerevisiae mitogen-activated protein kinase pathway-modulating protein STE50 and analysis of its interaction with the STE11 SAM. J Biol Chem 279: 2192–2201.
- 37. Truckses DM, Bloomekatz JE, Thorner J (2006) The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae. Mol Cell Biol 26: 912–928.
- 38. Wu C, Jansen G, Zhang J, Thomas DY, Whiteway M (2006) Adaptor protein Ste50p links the Ste11p MEKK to the HOG pathway through plasma membrane association. Genes Dev 20: 734–746.
- 39. Schamber A, Leroch M, Diwo J, Mendgen K, Hahn M (2010) The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea. Mol Plant Pathol 11: 105–119.
- 40. Park G, Xue C, Zhao X, Kim Y, Orbach M, et al. (2006) Multiple upstream signals converge on the adaptor protein Mst50 in Magnaporthe grisea. Plant Cell 18: 2822–2835.
- 41. Fu J, Mares C, Lizcano A, Liu Y, Wickes BL (2011) Insertional mutagenesis combined with an inducible filamentation phenotype reveals a conserved STE50 homologue in Cryptococcus neoformans that is required for monokaryotic fruiting and sexual reproduction. Mol Microbiol 79: 990–1007.
- 42. Bayram O, Bayram OS, Ahmed YL, Maruyama J, Valerius O, et al. (2012) The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. PLoS Genet 8: e1002816.
- 43. Pandey A, Roca MG, Read ND, Glass NL (2004) Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa. Eukaryot Cell 3: 348–358.
- 44. Maerz S, Ziv C, Vogt N, Helmstaedt K, Cohen N, et al. (2008) The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa. Genetics 179: 1313–1325.
- 45. Jamet-Vierny C, Debuchy R, Prigent M, Silar P (2007) IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina. Fungal Genet Biol 44: 1219–1230.
- 46. Belden WJ, Larrondo LF, Froehlich AC, Shi M, Chen CH, et al. (2007) The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output. Genes Dev 21: 1494–1505.
- 47. Heilig Y, Dettmann A, Mourino-Perez RR, Schmitt K, Valerius O, et al. (2014) Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1. PLoS Genet 10: e1004306.
- 48. Heilig Y, Schmitt K, Stephan Seiler (2013) S (2013) Phospho-regulation of the Neurospora crassa septation initiation network. PLOS One 8: e79464.
- 49. Kana-uchi A, Yamashiro CT, Tanabe S, Murayama T (1997) A ras homologue of Neurospora crassa regulates morphology. Mol Gen Genet 254: 427–432.
- 50. Roca MG, Arlt J, Jeffree CE, Read ND (2005) Cell biology of conidial anastomosis tubes in Neurospora crassa. Eukaryot Cell 4: 911–919.
- 51. Kramer B, Thines E, Foster AJ (2009) MAP kinase signalling pathway components and targets conserved between the distantly related plant pathogenic fungi Mycosphaerella graminicola and Magnaporthe grisea. Fungal Genet Biol 46: 667–681.
- 52. Dard N, Peter M (2006) Scaffold proteins in MAP kinase signaling: more than simple passive activating platforms. Bioessays 28: 146–156.
- 53. Witzel F, Maddison L, Bluthgen N (2012) How scaffolds shape MAPK signaling: what we know and opportunities for systems approaches. Front Physiol 3: 475.
- 54. Cote P, Sulea T, Dignard D, Wu C, Whiteway M (2011) Evolutionary reshaping of fungal mating pathway scaffold proteins. MBio 2: e00230–00210.
- 55. Jonkers W, Leeder AC, Ansong C, Yang F, Camp II DG, et al. (2014) HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa. PLoS Genet 10: e1004783.
- 56. Filippi BM, de los Heros P, Mehellou Y, Navratilova I, Gourlay R, et al. (2011) MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases. EMBO J 30: 1730–1741.
- 57. Gourlay CW, Ayscough KR (2006) Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae. Mol Cell Biol 26: 6487–6501.
- 58. Field J, Vojtek A, Ballester R, Bolger G, Colicelli J, et al. (1990) Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell 61: 319–327.
- 59. Zhou X, Zhang H, Li G, Shaw B, Xu JR (2012) The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae. PLoS Pathog 8: e1002911.
- 60. Rocha CR, Schroppel K, Harcus D, Marcil A, Dignard D, et al. (2001) Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 12: 3631–3643.
- 61. Bertling E, Quintero-Monzon O, Mattila PK, Goode BL, Lappalainen P (2007) Mechanism and biological role of profilin-Srv2/CAP interaction. J Cell Sci 120: 1225–1234.
- 62. Moriyama K, Yahara I (2002) Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. J Cell Sci 115: 1591–1601.
- 63. Lichius A, Goryachev AB, Fricker MD, Obara B, Castro-Longoria E, et al. (2014) CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa. J Cell Sci 127: 1953–1965.
- 64. Frieser SH, Hlubek A, Sandrock B, Bolker M (2011) Cla4 kinase triggers destruction of the Rac1-GEF Cdc24 during polarized growth in Ustilago maydis. Mol Biol Cell 22: 3253–3262.
- 65. Schuerg T, Brandt U, Adis C, Fleissner A (2012) The Saccharomyces cerevisiae BEM1 homologue in Neurospora crassa promotes co-ordinated cell behaviour resulting in cell fusion. Mol Microbiol 86: 349–366.
- 66. Araujo-Palomares CL, Richthammer C, Seiler S, Castro-Longoria E (2011) Functional characterization and cellular dynamics of the CDC-42 - RAC - CDC-24 module in Neurospora crassa. PLoS One 6: e27148.
- 67. Riquelme M, Yarden O, Bartnicki-Garcia S, Bowman B, Castro-Longoria E, et al. (2011) Architecture and development of the Neurospora crassa hypha — a model cell for polarized growth. Fungal Biol 115: 446–474.
- 68. Seiler S, Justa-Schuch D (2010) Conserved components, but distinct mechanisms for the placement and assembly of the cell division machinery in unicellular and filamentous ascomycetes. Mol Microbiol 78: 1058–1076.
- 69. Jedd G, Pieuchot L (2012) Multiple modes for gatekeeping at fungal cell-to-cell channels. Mol Microbiol 86: 1291–1294.
- 70. Heriche JK, Lebrin F, Rabilloud T, Leroy D, Chambaz EM, et al. (1997) Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha. Science 276: 952–955.
- 71. Lebrin F, Bianchini L, Rabilloud T, Chambaz EM, Goldberg Y (1999) CK2alpha-protein phosphatase 2A molecular complex: possible interaction with the MAP kinase pathway. Mol Cell Biochem 191: 207–212.
- 72. Goudreault M, D'Ambrosio LM, Kean MJ, Mullin MJ, Larsen BG, et al. (2009) A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics 8: 157–171.
- 73. Garrenton LS, Stefan CJ, McMurray MA, Emr SD, Thorner J (2010) Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling. Proc Natl Acad Sci U S A 107: 11805–11810.
- 74. Mahs A, Ischebeck T, Heilig Y, Stenzel I, Hempel F, et al. (2012) The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa. PLoS One 7: e51454.
- 75. Roelants FM, Baltz AG, Trott AE, Fereres S, Thorner J (2010) A protein kinase network regulates the function of aminophospholipid flippases. Proc Natl Acad Sci U S A 107: 34–39.
- 76. Nakano K, Yamamoto T, Kishimoto T, Noji T, Tanaka K (2008) Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry. Mol Biol Cell 19: 1783–1797.
- 77. Saito K, Fujimura-Kamada K, Hanamatsu H, Kato U, Umeda M, et al. (2007) Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins. Dev Cell 13: 743–751.
- 78. Kothe GO, Free SJ (1998) The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa. Genetics 149: 117–130.
- 79. Seiler S, Plamann M (2003) The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa. Mol Biol Cell 14: 4352–4364.
- 80. Freitag M, Hickey PC, Raju NB, Selker EU, Read ND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41: 897–910.
- 81. Honda S, Selker EU (2009) Tools for fungal proteomics: multifunctional neurospora vectors for gene replacement, protein expression and protein purification. Genetics 182: 11–23.
- 82. Verdin J, Bartnicki-Garcia S, Riquelme M (2009) Functional stratification of the Spitzenkorper of Neurospora crassa. Mol Microbiol 74: 1044–1053.
- 83. Maerz S, Dettmann A, Seiler S (2012) Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1. Mol Cell Biol 32: 2083–2098.
- 84. Seiler S, Vogt N, Ziv C, Gorovits R, Yarden O (2006) The STE20/germinal center kinase POD6 interacts with the NDR kinase COT1 and is involved in polar tip extension in Neurospora crassa. Mol Biol Cell 17: 4080–4092.
- 85. Richthammer C, Enseleit M, Sanchez-Leon E, Marz S, Heilig Y, et al. (2012) RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa. Mol Microbiol 85: 716–733.
- 86. Maddi A, Dettman A, Fu C, Seiler S, Free SJ (2012) WSC-1 and HAM-7 are MAK-1 MAP kinase pathway sensors required for cell wall integrity and hyphal fusion in Neurospora crassa. PLoS One 7: e42374.
- 87. Fu C, Ao J, Dettmann A, Seiler S, Free SJ (2014) Characterization of the Neurospora crassa cell fusion proteins HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2. PLoS ONE 9: e107773.
- 88. Riquelme M, Bredeweg EL, Callejas-Negrete O, Roberson RW, Ludwig S, et al. (2014) The Neurospora crassa exocyst complex tethers Spitzenkorper vesicles to the apical plasma membrane during polarized growth. Mol Biol Cell 25: 1312–1326.


text:
- First dates can be nerve-wracking.
- To understand each other, you people need to spend time as mentioned earlier.
- When it comes to what not to do on a first date, talking about yourself constantly is a big no-no.
This should be self-explanatory. Wondering what not to do on a first date? Shower, shave, brush your teeth, and change into clean clothes. You can always ask a friend to come with you and sit at another table, chilli dating ne if you don't feel safe going alone.
10 Golden Rules of A Relationship
Every relationship goes through a bad patch, but a few of them never split apart. We want to help our community find and shine their inner light - the truth of love, light, and positivity that is within us all! They always stay with you till the end. Take your own transportation.
These relationship rules will help you grow with your partner with the help of other factors like communication, understanding, intimacy, and loyalty. The most effective online dating profiles. And never speak over them.
Never order food for your date, unless he or she has expressly asked you to. Where was your partner headed before you met? Your date will often judge you more on your treatment of others than your treatment of him or her. Your mistakes, your achievements, your strengths, and your weaknesses, share with your partner.
There is always something which is not known to you about your partner. All the images are collected from Google. By communication, we mean listening more than speaking your meaning.
It could be a personal problem or a social issue that is affecting you. You and your partner will argue, but knowing how to fight fairly will be an important skill to keep your relationship solid through the years. Interact with each other to get closer.
If you are unsure whether to tell your partner about something, ask yourself if they found out another way if they would be upset. Something which is related to your relationship or something at work. Share your concern with your partner.
Instead, let your actions do the talking. Withholding information is likely to not win you any points with your partner either. But, when a healthy relationship is taken into consideration, then you people need to express what you think about your relationship and about your partner.
No one wants to be caged up, and the more freedom you can allow your partner to have, the greater their appreciation of the wide boundaries that you provide. Did you derail them from reaching their goals? Even if the other person has the same aims as you, bringing them up within minutes of meeting is only likely to scare him or her away. Try to live every moment of your life with each other to understand which is the real personality of your partner. Be the best friend and companion to your partner as well as a romantic mate.
The 10 Golden Rules Of Sugar Dating (From A Real Life Sugar Baby)
Facebook Pinterest Twitter YouTube. Know their love language and give them love in the way in which they like to receive it. Today is not where you will be ten years from now. No matter how great he sounds, insist on meeting in public for your initial meeting, and even your second or third meeting. You rarely get second chances, b2 canada dating so make sure you feel at your best.
10 Important Ground Rules For A New Relationship
Flat out lying is definitely frowned upon. You need to understand that every person needs space once in a while to breathe, dating to live their life in their own way. Intimacy helps you to come closer but it never helps you to understand each other. It also takes away some of those first-date jitters by knowing this isn't really a date.
Try eHarmony for free today! They probably have not forgotten what they dreamed of before they met you, so be supportive of their future goals as well as your own. Patience and tolerance are two basic of a relationship to keep you two together. Challenge your relationship to strengthen your bond with each other.
If your partner is interested in something, they are basically asking you to turn toward them. We are different individuals and we have our individual opinions. Avoid discussing anything too serious or confrontational. Spending time with each other will help you to understand each other in a deeper manner.
Be patient and try to calmlytackle a critical situation. It can also be difficult for people to accurately represent their feelings with a total stranger. If this article gave you the confidence to find your match, try eHarmony today! No matter how your last relationship ended, avoid talking about it on a first date.
Do you want to have a healthy long-term relationship? Our passion is to serve and bring the best possible positive information, news, expertise and opinions to this page. And remember, never leave your drink unattended on a date with a stranger.
Here Are 10 Golden Rules Every Relationship Needs To Know About
If something doesn't seem right before you meet, don't do it. On the other hand, another study found that power imbalances and unequal treatment was a reason that women more often cited for breakups than men did. Just spending your time with your partner is not enough. Hold doors open, say please and thank you, and eat with your mouth closed.
More From Thought Catalog
Do not force your partner to spend every bit of his or her life with you. Power of Positivity Our passion is to serve and bring the best possible positive information, news, expertise and opinions to this page. Don't be afraid to have fun! Be as supportive as possible to your partner for this golden rule, or your partner may find the support elsewhere. Do not try to lie, just speak straight to the point.
Do your very best to follow this golden rule that every relationship needs to know because the consequences of being less than honest are lonely and painful. Related Items dating golden rules in relationships love relationship advice relationships romance. There are a few golden rules of a relationship, one should follow to keep your relationship intact for years and years. So, follow these rules to make your relationship a stronger one and let us know if there are any other factor which is left untouched by us.
The 10 Golden Rules Of Sugar Dating (From A Real Life Sugar Baby)
The end result of sharing your memories with your partner will come as a strengthen bond between you two. Your first meeting doesn't count as a first date. There are a number of things you can do to make your relationship stronger. Let it go or the weight of that baggage will drag your relationship down. First impressions are important.
The 10 Golden Rules Of Online Dating No Cheating
1. Fight fair
Check in with your partner to see how they feel about the power balance in your relationship. Let your partner know that you love him or her from the bottom of your heart. You can find many ways to deal with a situation when your mind is open enough to look at the problem with several different perspectives. Compatibility criteria apply.
- Power of Positivity uses cookies to help us provide, protect and improve our site.
- If you decide to turn away, you deny their request for your attention.
- We all crave love, but not just any love.
- Provide what your partner likes and needs.
- Do not ever impose yourself on your partner as it will surely affect your relationship very badly.

subset: commoncrawl


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John Mehegan
|June 6, 1916
Hartford, Connecticut, U.S.
|April 3, 1984 (aged 67)
New Canaan, Connecticut
|Musician, lecturer, critic
John Francis Mehegan (June 6, 1916 – April 3, 1984) was an American jazz pianist, lecturer and critic.
Early life
Mehegan was born in Hartford, Connecticut, on June 6, 1916, although he sometimes gave the year as 1920.[1] He began playing the violin in 1926 and played for seven years without enjoying it.[1] He initially taught himself to play the piano by matching his fingers to the notes played on a player piano.[1] He went on to study at the Hartt School of Music in Hartford.[1] He had gigs in the Massachusetts area, and then moved to New York in 1941.[2]
Later life and career
In New York, Mehegan played in clubs.[1] He recorded four quartet tracks as a leader for Savoy Records in 1945.[1][2] In the same year, he became teaching assistant to pianist Teddy Wilson in the jazz department at the Metropolitan Music School,[3] and became the head of its jazz department in 1946; a position he held for around a decade.[1] He also taught at the Juilliard School of Music (1947–64), Columbia University Teachers College (1958 to 1961 or 1962), the University of Bridgeport (1968–77) and Yale University (1974–83).[1]
He wrote the incidental music for A Streetcar Named Desire which he performed on Broadway for two years.[3] Mehegan was questioned by the House Un-American Activities Committee, where he was an uncooperative witness.[4] He was the jazz critic for the New York Herald Tribune from 1957 to 1960.[3] "A summer concert, lecture, and research tour of South Africa in 1959 was cut short because he encouraged black musicians, but while there he recorded with the group which was about to become the Jazz Epistles".[1] His final recordings, as a trio, were made in 1960.[2]
He wrote numerous books on jazz, including the Jazz Improvisation series, which sets out the basic principles of jazz,[5] and was published between 1959 and 1965.[1]
The American composer Leonard Bernstein dedicated a piano composition to Mehegan in his 1948 collection Four Anniversaries.[6] Mehegan died in New Canaan, Connecticut, on April 3, 1984.[1]
- 1952 From Barrelhouse to Bop (Perspective) - with Charles Mingus
- 1954 The First Mehegan (Savoy)
- 1955 The John Mehegan Trio/Quartet (Savoy) - with Charles Mingus & Kenny Clarke
- 1955 A Pair of Pianos (Savoy)- with Eddie Costa & Vinnie Burke
- 1956 How I Play Jazz Piano (Savoy)
- 1959 Casual Affair
With Chuck Wayne
- The Jazz Guitarist (Savoy, 1954 [1956])
Techniques, studies & etudes for piano
- Contemporary Styles for the Jazz Pianist, in 3 books (1964–70)
- Famous Jazz Style Piano Folio - with instruction on how to play jazz piano (1958)
- Jazz Improvisation (1959-65)
- Vol. 1: Tonal and rhythmic principles (1959)
- Vol. 2: Jazz rhythm and the improvised line (1962)
- Vol. 3: Swing and early progressive piano styles (1964)
- Vol. 4: Contemporary piano styles (1965)
- The Jazz Pianist, in 3 books: Studies in the art and practice of jazz improvisation (1960–61)
- Styles for the Jazz Pianist, in 3 books (1962–63)
- Studies in Jazz Harmony (1962)
Original compositions for piano
- Jazz Bourree (1960)
- Jazz Preludes (1962)
- Vienna Woodshed, a jazz waltz for piano 4-hands (1965)
- Jazz Caper, jazz originals for piano 4-hands (1965)
- ^ a b c d e f g h i j k Kernfeld, Barry (2003), Mehegan, John (Francis), Grove Music Online. Oxford Music Online. Oxford University Press, retrieved December 19, 2018
- ^ a b c Yanow, Scott. "John Mehegan". AllMusic. Retrieved December 19, 2018.
- ^ a b c Wilson, John S. (April 5, 1984) "John Mehegan, Jazz Pianist; Wrote 4-Volume Textbook" New York Times p D30
- ^ Porter, Russell (April 10, 1957) "Inquiry Charges Red Link in Music" New York Times, p 18L
- ^ Dunscomb, Richard J. & Hill Jr., Willie L. (2002) Jazz Pedagogy Alfred Music Publishing, p336
- ^ Allmusic


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Scientists are hailing this prehistoric dinosaur as the "best-preserved dinosaur on Earth." In fact, it is so well preserved that it cannot be defined as a fossil. This magnificent ancient 18-foot-long specimen has been called a genuine "dinosaur mummy."
تم النشر بواسطة Royal Tyrrell Museum of Palaeontology في الجمعة، ١٠ مايو ٢٠١٩
On May 12, 2017, the Royal Tyrrell Museum of Palaeontology in Alberta, Canada, unveiled a dinosaur exhibit: "We don't just have a skeleton," Caleb Brown, a researcher at the museum, told National Geographic. "We have a dinosaur as it would have been."
Since it was unearthed, it has kept its shape; its bones aren't visible and even some of its innards are still intact. Researchers were amazed at the extent of the almost unparalleled degree of preservation.
The dinosaur is so well preserved that it "might have been walking around a couple of weeks ago," Jakob Vinther, a paleobiologist from the University of Bristol, said. "I've never seen anything like this."
تم النشر بواسطة Royal Tyrrell Museum of Palaeontology في الخميس، ٣ أغسطس ٢٠١٧
The creature was first discovered in 2011 when an oil mine employee named Shawn Funk inadvertently discovered the specimen while at work.
While Funk was excavating that day, he was surprised to find something he had never discovered in 12 years of digging. In the afternoon, Funk's excavator's claw struck something different. He and his supervisor, Mike Gratton, started to wonder what these strange-colored lumps were. They questioned if these were fossilized wood or an animal's ribs?
"Right away, Mike was like, 'We gotta get this checked out,'" Funk said in a 2011 interview. "It was definitely nothing we had ever seen before." Surprisingly, this wasn't fossilized wood or a petrified tree stump but a fossilized dinosaur.
From its finding, it took researchers six years and around 7,000 hours to conduct tests on the evidence collected and prepare the remnants to be displayed at the Royal Tyrrell Museum of Palaeontology.
A remarkably intact dinosaur glares back at us, thanks to a long-vanished sea in Alberta.
تم النشر بواسطة The New York Times في الجمعة، ١٢ مايو ٢٠١٧
Over 100 million years ago, when this ancient prehistoric creature roamed the earth, it was a member of a newly discovered species and genus named the Nodosaur.
According to paleontologists, this bizarre fossil was the first of its kind. Usually, it's rare to find a fossil that keeps its soft tissue in its true shape. It's common to see bones and teeth well preserved. So indeed, this was an amazing discovery.
It was a gigantic four-legged herbivore, and its body was protected by spiky, plated "armor." This nodosaur originally weighed around 3,000 pounds (approx. 1,361 kg) when it was alive. The mummified nodosaur is so unimpaired that it still weighs 2,500 pounds (approx. 1,134 kg)! Truly marvelous!
When the museum put up pictures of the dinosaur on their Facebook page, one curious user commented: "What was it's favorite plant to eat? Was it prone to calm then hissing and snapping like a turtle when provoked, or was it's demeanor different? Can you tell if it's male or female?"
Join Dr. Henderson on @CTVCalgary today at noon for insights on some of the amazing specimens in our new exhibit Grounds for Discovery.
تم النشر بواسطة Royal Tyrrell Museum of Palaeontology في الخميس، ٣١ أغسطس ٢٠١٧
To which Brown replied: "1) We don't know what its favorite plant food was. We do know that it would have enjoyed plants growing close to the ground, as it would not have been able to reach very high up. Hopefully, analysis of the stomach contents will allow us to determine the last plants that it ate. 2) We don't know what its demeanor was like. It would have probably been slow moving, but we are unsure if it was a gentle giant or was defensive, snapping creature. 3) We also don't know if it was male or female. Outside of finding eggs inside the animal, there are few ways to tell male and female dinosaurs apart. Although the skin is preserved on much of the animal, we don't have the pelvic region preserved, so we also can't see any structures that may have been preserve there. It is interesting to consider what may have been preserved had that region been recovered."
The question of how the dinosaur could have remained so intact after it was unearthed still baffles scientists, although according to National Geographic, researchers are putting forth the theory that the creature may have been carried away to sea by a "flooded river" where it sank. As it lay there undisturbed, the armor and skin were replaced by minerals over the course of millions of years. This gave it the form that it is currently in, on exhibit.
تم النشر بواسطة Royal Tyrrell Museum of Palaeontology في الخميس، ٣ أغسطس ٢٠١٧
Alluding to this amazing find, Brown said: "It will go down in science history as one of the most beautiful and best-preserved dinosaur specimens—the Mona Lisa of dinosaurs."
With the dinosaur mummy on display at the Royal Tyrrell Museum, visitors have an opportunity to observe what is considered to be the closest thing to an original dinosaur. On seeing this amazing specimen, perhaps visitors will be able to vividly visualize the lives and times of dinosaurs, so many millions of years ago.


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Kabbalah is the soul of Torah. Kabbalah is the study of the soul: The human soul, and the soul of Torah, and the soul of everything in the material world.
"There are many layers to reality. The material world experience as we know it is just the surface level, within which exist many dimensions of spiritual energy waiting to be released. By appreciating these inner layers, we can learn to make order out of disorder, to gain insight into the unknown forces that determine all movement and behavior." — From Toward a Meaningful Life by Rabbi Simon Jacobson
Why Men Make Kiddush and Women Light Shabbat Candles
Since according to basic Jewish law (halacha), a woman can make kiddush and a man can light Shabbos candles, why is the custom for women to light the candles?Read More
The Kabbalah of the Big Bang
Does the Big Bang Theory have its roots in religious and mystical teachings? Or, as some have argued, has it deemed the notion of a Creator superfluous?Read More
Three Weeks of Pain, Seven of Comfort, & Two of Return
Cracks in our systems shake us up; but a crack also exposes what lies within. We can be demoralized by the cracks or we can attempt to look between the cracks and see an emerging larger picture.Read More
Kabbalah: Uniting Heaven & Earth
The Kabbalah (Jewish mysticism), and specifically the branch known as Chassidus, addresses the "cosmic order," which unites spirit and matter. This is an excerpt from "Toward a Meaningful Life".Read More
What Is Kabbalah?
Kabbalah is the study of the soul: The human soul, and the soul of Torah, and the soul of everything in the material world. Find out what is Kabbalah and Kabbalism from a renowned Chassidic master teacher, Rabbi Simon Jacobson.Read More
Kabbalah: The Inner Journey
Kabbalah is a word in Hebrew that means literally, "reception," to receive. This refers to the reception of student from teacher in a long, unbroken chain of esoteric teachings of Jewish mysticism, the most classical text of Kabbalah known to most people being the Zohar, authored by Rabbi Shimon bar Yochai who lived approximately 2,000 years ago.Read More
Your Seven Emotions
In the Kabbalah, Jewish mysticism, there is a map of the emotions. It's a map that divides the emotions into seven categories, seven particular attributes and faculties.Read More
Kabbalah of Chanukah
Each of the seven candles represents one of the emotional characteristics listed in the Zohar (the main book of the Kabbalah).Read More
The Kabbalah of Curses
The 49 curses listed in the Torah portion of Bechukosei evokes an interesting question: what possible benefit can be found in these curses?Read More
Re'eh: The Kabbalah of Duality
Part II of the series asks important questions about how we reconcile faith and modernity and discusses how spirituality is relevant in contemporary times..Read More


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29 Fentoun Gait East, GULLANE, EH31 2ES
Fixed Price £699,000 | 5 Bed House - Detached with 3 Reception Rooms
Ref: E406210
The Stevenson fully embraces modern, flexible living. From its large lounge with double doors, through to the elegant rear dining room and into the extensive open plan kitchen/living area, which boasts bi-folding doors to the garden and a separate utility room. They are joined by private study, as well as a cloakroom and plentiful storage throughout. All five bedrooms include fitted wardrobes, with the master and second bedrooms also enjoying a luxury en suite and fitted wardrobes. Further complemented with a four-piece family bathroom and detached double garage outside. Overall size: 2,399 sq.ft
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Tel: 01620 807624
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Pinsent Masons LLP - EDINBURGH
1 Earl Grey Street, Princes Exchange, Edinburgh, EH3 9AQ
Tel: 0131 253 2177
Fax: 0131 777 7003
Web: http://www.pinsentmasons.com/


text:
Here's a follow up to part one of the Bare Essentials for Nude Camping Interstate on a Budget. If you value your sleep and don't want an interrupted rest, here's three more optional extras you may want to consider adding to the list.Read More
You may think that nude camping wouldn't require much planning. After all, clothes are the majority of the packing right? Before you jump on a plane and head off on a spontaneous nude camping trip, here's a few things I had to consider that may be useful to know.Read More
If you're the sort to linger a little longer stepping starkers out of the shower, or you are wanting to progress your body positivity to peak performance. Consider trying one of these nude (and certainly not rude) Australian events this summer.Read More


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Fitness And Health
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Garrucha, 19, 04140 Carboneras, Spain Show on the map
Choose your room
Located in the town of Carboneras, within the Natural Park Cabo de Gata-Níjar, and a few minutes from the most impressive beaches in southern Europe. We have 25 rooms, the vast majority with terrace, their decoration and equipment offer a magnificent result: elegant, modern and comfortable. The rooms combine perfectly with the common areas of the hotel, following a discreet and elegant line. The bathrooms, equipped with hairdryer and quality amenities, are decorated in a simple and practical way.
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found here. It's important for you to consider the current Financial Service Guide (FSG), Product Disclosure Statement PDS, Account Terms and any other relevant oanda documents before making any financial investment decisions.
Christmas trading hours @ Forex Factory Bitcoins: the Complete Guide: 12 Steps
Oanda Australia Pty Ltd is regulated by the Australian Securities and Investments Commission asic (ABN, afsl. Spot Gold and Silver Market, hours. These bitcoin mining step by step pdf changes will take place as follows: Christmas : Monday 24 December - Wednesday 26 December. This is for general information purposes only - Examples shown are for illustrative purposes and may not reflect current prices from oanda. Refer to our legal section here. WTI, closes 20:30, closed. Spot gold and silver trading also follow, cME holiday closures. Leverage trading is high risk and not for everyone. Forex, market, hours, forex, trading is available 24 hours a day from 5:00pm ET Sunday through 5:00pm ET on Friday, including most.S. National bank holidays and weekends are taken into account. Oanda uses cookies to make our websites easy to use and customized to our visitors. Kind regards, Admiral Markets.
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Things to do in Orta San Giulio
Top recommendations from locals
From sightseeing to hidden gems, find out what makes the city unique with the help of the locals who know it best.
Italian Restaurant
"A two star Michelain restaurant set in a magnificent Villa at the south of the lake. A very special place serving excellent food with professional staff and attention to detail. It is expensive but..."
- 10 locals recommend
"Orta San Giulio is a piece of Middle Age. Nice shopping, romantic promanades, often proposing street markets"
- 60 locals recommend
"It's a UNESCO Heritage Site (since 2003!), incredibly panoramic and well preserved, encompassing 21 buildings fully frescoed and embellished with multicolored terracotta statues dating, all dating back mainly to the 17th century! The site is surrounded by a Nature Reserve in which you can stroll and relax, enjoying the breathtaking view of Lake Orta. You can reach it from Orta car-parking in 20 minutes on foot."
- 5 locals recommend
"Ristorante sul lago che serve carne, pesce e pizza, oltre ad aperitivi, in un contesto elegante e raffinato."
- 3 locals recommend
"E' il terzo birrificio in italia per il numero di tipologie di birre a disposizione degli ospiti"
- 1 local recommends
"An eclectic owner who uses her designer talents to modify the ambience regularly, excellent fresh food, great wine also by the glass, good prices, the friendliest waiters in town, one of my regular spots. "
- 3 locals recommend
"Sehr gutes, original piemontesisches Restaurant mit frischen Produkten aus eigenem Anbau nach der Slow-Food-Philosophie. Man folgt am besten der Empfehlung der Gastgeberin und muss oft bereits nach dem Primo passen, damit noch Platz fürs Dolce bleibt! Unbedingt reservieren. Sonntagmittag bei italienischen Familien sehr beliebt. Ristorante Maria Piera +39 339/5775385 www.ilcucchiaiodilegno.com"
- 1 local recommends
Pizza Place
"Ottima vista e menu, buon rapporto qualità/prezzo. D'estate molto bella la terrazza con vista sull'isola di S. Giulio."
- 1 local recommends
Place of Worship
"A nice drive to get here and interesting visit. It is a peninsula jutting out into the lake, scattered with little chapels each one different to the other. Its position gives you a splendid view over the lake towards the little island of San Giulio. There is something quiet magical about this little known lake. Omegna is the largest town on Orta and has a market on Thursdays."
- 5 locals recommend
"A new location hidden away on the left up the cobbled road leading to the main church. Lovely old style location on different floors, small and homely. Good food and wine. "
- 1 local recommends
"Newly opened gourmet restaurant with Michelin one star chef Fabrizio Tesse. Well worth a visit for his multiple antipasti creations. "
- 2 locals recommend
"A good restaurant where the food is fresh, the wine excellent and they cater to all requests with punctual and friendly service. Their dining saloon upstairs offers a spectacular view of the island. They also do good pizza & cocktails."
- 3 locals recommend


text:
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MS Roofing Supplies
Metal roofing is much stronger and more durable than asphalt shingle roofing. With Custom Metal Solutions 40 year limited paint warranty on our panels, your metal roof can last twice as long as most conventional roofs. Because metal roofing is so durable, it has long been the choice for many Military, School, Government, and Commercial building applications.
Metal roofing is superior to asphalt roofing products in all types of weather conditions, including hail, sun exposure, and wind. Unlike asphalt shingles, metal roofing will not deteriorate due to sun exposure. High winds and/or a hail storm will destroy an asphalt roof, but a roof from Custom Metal Solutions can handle majority of the wind speeds experienced here in Central Mississippi area. Our products are also extremely hail resistant. Most insurance companies in our area offer a discount on your homeowners insurance for a new metal roof installation. Percentages may vary so please verify with your insurance carrier for details.
Jackson Sheet Metal
Lighter colored metal roofs reflect away much of the sun's UV rays, which can help keep your attic space in your home cooler and lower cooling costs. In recent studies done in Florida, a metal roof can cut cooling costs by as much as 25% or more.
Our paint systems are top of the line. We use ceramic pigments that incorporate "Cool Chemistry" technology. This technology keeps your roof cooler, helping you save on your energy costs. Our panels have superior color retention. They also stand up better to scratches and abrasions that occur from objects hitting the roof panels. To learn more about our paint systems, visit the Color Selector section of our website.
Construction Jackson MS
According to the National Association of Homebuilders Research Center, 20 billion pounds of asphalt shingles are dumped into U.S. landfills annually. The recycled content of steel in metal roofing is much higher than the recycled content in asphalt shingles. Also, due to the low weight of metal roofing (about half that of shingles), in many cases it can be installed directly over your existing roof, saving the environmental as well as financial costs associated with removal and disposal.
The following are the qualities of certified roof specialists:.
MS Roofing Service provider Professionals is everything about services beyond the proprietors' expectations. It is cost reliable, a reputable and reliable business among the couple of proficient roofer Jackson, MS. It is a great opportunity to provide your home the very best roofing in Jackson, MS.
We meet all your roofing needs at roofing contractors Jackson, MS. Roofing Jackson, MS will make sure that you have an awesome roof repair experience. Other roofing contractors many times don't give you a good price on your estimate because they assume that you know anything about roofing. They use this to manipulate you into paying more money that you should. Roofing Contractors Jackson, MS we believe in a few principles to make the best decision possible. So our qualified roofers:
1. Educate you about your problem
2. Suggest most economical Solutions
3. Provide excellent customer service completing Repair
Give us a call if you are looking for a great roofer in Jackson, MS.
• Full Service Roofing
• Emergency Roof Repair
• New Roofs
• Re-Roofs
• Residential & Commercial
• Gutter Installs
• Tile, Shingle, or Flat Roof Service
• Jackson Roofing Leak Repair
• Jackson Metal Roofing
The following are the qualities of certified roof specialists:.
• Specialized Professionals.
o Each Roofer Professionals' professional is an expert, in reality a qualified expert experienced in their work. For that reason, they provide accommodation to all roof requirements. All the professionals are masterfully trained and have actually done and passed in their occupation training. The specialists take pride in long years of practice in the industry.
• Exceptional Customer Service
o All proprietors are on the search for laborers who are capable, skilled, and able to deal with all roof jobs. In addition, technicians who prioritize in excellent customer service.
• Outstanding Abilities
o Given that a roof over a residence is the most important security for the occupant, it should be cared for. Bearing in mind that a shoddily installed roof can really because more harm than good. It is essential to have a professional with professional abilities.
• Well-timed Roofing Service
o Home owners require specialists who will perform the roof assignment in a period required.
Roofing Service provider Professionals is everything about services beyond the proprietors' expectations. It is cost reliable, a reputable and reliable business among the couple of proficient roofer Jackson, MS. It is a great opportunity to provide your home the very best roofing in Jackson, MS.