In contrast, vertically adjacent sections of the sediment laden basal ice contained much higher concentrations of CO2 , whereas O2 represented 4 to 18% of the total gas volume. The deviation in gas composition from atmospheric values occurred concurrently with increased microbial cell concentrations in the basal ice profile, suggesting that in situ microbial processes  may have altered the entrapped gas composition. Molecular characterization of 16S rRNA genes amplified from samples of the basal ice indicated a low diversity of bacteria, and most of the sequences characterized (87%) were affiliated with the phylum, Firmicutes. The most abundant phylotypes in libraries from ice horizons with elevated CO2 and depleted O2 concentrations were related to the genus Paenisporosarcina, and 28 isolates from this genus were obtained by enrichment culturing. Metabolic experiments with Paenisporosarcina sp. TG14 revealed its capacity to conduct macromolecular synthesis when frozen in water derived from melted basal ice samples and incubated at −15 °C. The results support the hypothesis that the basal ice of glaciers and ice sheets are cryospheric habitats harboring bacteria with the physiological capacity to remain metabolically active and biogeochemically cycle elements within the subglacial environment.Measurement of gases entrapped in clean ice from basal portions of the Taylor Glacier, Antarctica, revealed that CO Killed controls were amended with ice-cold 50% trichloroacetic acid (TCA) to a final concentration of 7% (w/v) 30 min prior to the addition of [3H]-leucine or [3H]-thymidine. All solutions were maintained on ice, and the samples were placed in a −80 °C freezer within 30s after the addition of either [3H]-leucine or [3H]-thymidine. After overnight (16 h) incubation at −80 °C, samples were transferred to a −15 °C thermally stable freezer (Revco ULT350-3-A32), which was designated as time zero. A HOBO U12 (Onset) data logger was used to log the temperature in the freezer every 10 min. Over the entire experimental time course, the mean temperature was −15.0 ± 0.5 °C. At each experimental time-point, frozen samples were removed from the freezer, immediately overlain with 100 μL of ice-cold 50% TCA, briefly centrifuged (10 s), and incubated at 4 °C to allow melting . After at least 30 min, the acid-insoluble macromolecules were pelleted by centrifugation at 17,000 × g for 15 min; the pellet was rinsed with 1 mL of ice-cold 5% TCA and centrifuged at 17,000 × g for 5 min. The residual was rinsed with 1 mL of ice-cold 70% ethanol, centrifuged at 17,000 × g for 5 min and the supernatant removed. The rinsed pellet was suspended in 1 mL of Cytoscint scintillation cocktail , and the radioactivity present was quantified using liquid scintillation spectrometry (Beckman LS6000IC scintillation counter). The number of disintegrations per minute (DPM) was calculated by determining counting efficiency using acetone-quenched standards of [3H] toluene  in the Cytoscint cocktail. Incorporation rates per CFU were calculated over time and converted to molecules of substrate incorporated, per CFU, per day (molecules CFU−1 day−1). Rate measurements were converted to grams of substrate carbon incorporated, per gram of cell carbon, per day (gC gC−1 day−1) based on 65 fg C cell−1 . In the debris-rich banded dispersed ice (profile depth 220 cm to 240 cm), total cell concentrations were approximately one to two orders of magnitude higher than the clean ice. SYBR Gold counts ranged from 1.8 ± 0.1 × 103 cells g−1 to 1.8 ± 0.4 × 104 cells g−1, while Baclight counts ranged from 2.4 ± 0.1 × 103 cells g−1 to 1.6 ± 0.3 × 104 cells g−1 (r(14) = 0.60, p < 0.05] and the concentration of CO2  and O2 , whose concentrations were elevated and depleted, respectively, relative to atmospheric values -leucine and [3H]-thymidine into acid-insoluble macromolecules when incubated at −15 °C in frozen melt water from either the clean or sediment-containing ice  over the entire time-course. For the banded dispersed ice melt water, incorporation of [3H]-leucine was initially rapid, but high variability in the data resulted in a poor fit of the regression model (α = 0.05); however, [3H]-thymidine incorporation appeared to be partially hyperbolic  and [3H]-thymidine (p > 0.79) incorporation measured in the clean ice and banded dispersed ice. However, cells in the clean ice incorporated significantly more [3H]-leucine (p = 0.049) and [3H]-thymidine (p = 0.003) than incubations conducted in the banded dispersed ice over 70 days.Isolate ning ice . During i.e., through regelation) from subglacial sources into the basal zone of the ice mass [Glaciers and ice sheets move under their own weight, entraining sediment particles and liquid water  with a decrease of O2  with respect to atmospheric concentrations (r (6) = −0.92, p < 0.005; 2 and O2 concentrations may have resulted from the aerobic respiration of organic matter. Indeed, the phylotypes identified in this study were related to predominantly aerobic microorganisms, and only two genera, Bacillus and Paenibacillus, are known to contain facultatively anaerobic species. These observations are similar to those in other basal ice environments, where similar gas anomalies have been reported. For instance, Souchez et al. [2  and CH4  concentrations in basal ice from the Greenland ice sheet. Likewise, Campen et al. [2, N2O and CH4  in ice from the Sajama glacier (Bolivia) and argued they resulted from microbial activity in the ice.Analysis of banded dispersed basal horizons from Taylor Glacier revealed a simultaneous increase of COz et al.  reportedn et al.  found exMolecular analysis of 16S rRNA genes amplified from Taylor Glacier basal ice revealed a low diversity of phylotypes from two bacterial phyla: the Firmicutes and Gammaproteobacteria. Although microorganisms attached to sediment particles in the basal ice may have been underrepresented in the 2009 samples, due to the sediment extraction procedure used, OTU diversity in the 2007 and 2009 was similar . The diversity of OTUs in the banded dispersed basal ice (H’ = ~1.8) was significantly lower than that reported for Arctic permafrost  and simPaenisporosarcina  along the north margin of the Taylor Glacier. Some members of the Firmicutes, including species of Paenisporosarcina , have the ability to differentiate into an endospore that is highly resistant to variety of environmental stresses. As such, their abundance in the ice could be attributed to selection, whereupon cells existing as environmentally durable spores would be more likely to persist in the ice after entrainment. However, results from pasteurization experiments indicated that 96% of the CFU were sensitive to heating at 80 °C, suggesting that the vast majority of the isolates obtained did not originate from heat resistant spores. The most abundant phylotypes in the debris-rich ice horizons were affiliated with the genus, osarcina . Enrichmasal ice . WhetherPaenisporosarcina have been documented in a variety of permanently cold environments, including permafrost [Paenisporosarcina-related isolates from this study exhibited moderate halotolerance between 6% and 12% (w/v). These levels of halotolerance do not appear to be sufficient for the estimated salt concentrations occurring inside Taylor Glacier basal ice veins  once every ~20 years. The annual N and P requirement to support this rate of DNA and protein synthesis in the TG14 cell population is estimated at approximately 2 ng N y−1 and 50 fg P y−1. In terms of carbon turnover, these metabolic rates are extremely slow (0.81 to 2.1 × 10−7 gC gC−1 h−1), but would be sufficient for a maintenance metabolism at −15 °C directed towards sustaining vital cellular processes  [Experiments were conducted in an effort to examine bacterial metabolism under conditions that simulated those within the basal ice. To ensure minimal carry-over of nutrients from the marine media, the harvested cells were washed and suspended in Taylor Glacier melt water. Marine broth contains 1,800 μg Llean ice . Paenisplecules) . −1, 0.68 mg L−1 and 5.5 μg L−1 respectively. These concentrations are approximately 102-fold higher than those measured in the debris-poor clean ice [Paenisporosarcina sp. TG14 at −15 °C, as compared with cells frozen in the clean ice  would be smaller than those in ice formed from more concentrated solutions . The unfrozen water volume of the clean ice is significantly smaller than that of the banded dispersed ice based on bulk concentration differences , and do not account for heterogeneity across a volume of ice.From the melted debris-rich basal ice (230–235 cm depth), the highest measured concentrations of dissolved organic carbon, dissolved inorganic nitrogen and dissolved inorganic phosphorus were 63 mg Llean ice . Furtherlk phase . MathemaPaenisporosarcina, were a numerically abundant member of this assemblage and appear to have characteristics that would promote survival in ice (e.g., psychrotolerance, halotolerance, metabolic activity to temperatures as low as −33 °C [In summary, basal horizons of the Taylor Glacier contained elevated concentrations of microbial cells, which consisted largely of bacteria in the phylum, Firmicutes. Species from the genus, s −33 °C  and a sps −33 °C .


During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients. During mitosis, the condensed chromosomes segregate into two genetically identical daughter cells so that each daughter cell contains a full set of chromosomes and a single centrosome. Chromosome segregation is carried out by the mitotic spindle, a dynamic molecular machine formed from microtubules and hundreds of other proteins. The fidelity of chromosome segregation is enhanced by cell-cycle checkpoints such as the spindle assembly checkpoint (SAC), which monitors the assembly of the mitotic spindle and delays chromosome segregation until every chromosome is correctly attached to spindle microtubules via kinetochores . MitoticThe activities of mitotic kinases are regulated in both space and time by post-translational modifications and protein–protein interactions. The most common forms of post-translational modification are phosphorylation  and ubiquitination . Protein-binding partners can either stimulate or activate kinase activity, and can specify the subcellular localization of a population of the kinase. Unravelling these mechanisms of regulation will be crucial for understanding the aetiology of some human diseases and developing new treatments for patients. The regulatory mechanisms of these kinases have previously been compared individually with those of the archetypal examples such as protein kinase A (PKA) or cyclin-dependent kinases (Cdks). We feel that a cross-comparison of mitotic kinases will be helpful for those in the mitosis field, and to highlight the ways in which these kinases diverge from archetypal kinases.In this review, we appraise the available structural information on the mitotic kinases and describe how this has revealed mechanisms of regulation at the molecular level. There are many protein kinases that influence mitotic events, but for reasons of brevity and clarity, this review will focus on the subset of kinase families that have members that are generally agreed to carry out their main function during mitosis, and for which there are available crystal structures: Cdk, Plk, Aurora, Nek, Bub, Haspin and Mps1/TTK. We will describe the crystal structures that have been determined, summarize the important conserved structural features that are crucial for activity and activation, and explain the key mechanisms of their regulation .Table 13.There are 518 known protein kinases in the human kinome that are divided by primary sequence into seven groups . With th3.1.Cdks are a large family of serine/threonine kinases that have been well studied since their discovery in the 1970s . Cdks beSaccharomyces cerevisiae (cdc28) and in Schizosaccharomyces pombe (cdc2), and is the key regulator of the G2/M transition, when it is subject to control by the DNA damage checkpoint . I. Ib) –80c) . T. Tb) [827.Many protein kinases require phosphorylation for activation, at one or multiple serine, threonine and tyrosine residues. Although phosphorylation can occur at sites throughout the protein, the region that is most commonly involved in regulation is the activation loop. Phosphorylation here is often a key characteristic of protein kinase regulation and is frequently used as a marker of kinase activity. The activation loops of kinases vary in length and often contain multiple serine, threonine or tyrosine residues, making it difficult to predict the sites of activating phosphorylation based purely on sequence . PhosphoKinases that are activated through phosphorylation of their activation loop usually have a canonical HRD motif, and the arginine residue within the motif recognizes the phosphorylated activation loop . This ina), pThr160 is mostly buried, where it interacts with three arginine residues at the canonical positions .Phosphorylation of Cdk2/Cyclin A complex is essential for full activity, although because CAK is constitutively active, this is not a regulatory step in the strictest sense. In the structure of the phosphorylated Cdk2/Cyclin A complex a, pThr16c and There is ongoing debate regarding the kinases upstream of Plk1, which include Aurora-A, Aurora-B, Plkk1  and PKA ,87–90. Tfigure 7e,f). The final stabilization of the activation segment is brought about by binding of TPX2, which will be described in the following section. There is no structure of phosphorylated Aurora-B alone, so it is not clear whether phosphorylation is sufficient to order the activation segment. Aurora-A has also been reported to be activated by phosphorylation at multiple sites within its N-terminal domain, which has been observed as an autoinhibitory domain [Aurora-A and Aurora-B kinase are phosphorylated at threonine residues within their activation loops at Thr288 and Thr232, respectively, which increases catalytic activity ,86. Althfigure 7y domain –97. HoweNek6 and Ser195Nek7, respectively [Plk1/Thr288Aurora-A, and they all have at least two basic residues in the canonical positions for phosphate recognition that could stabilize an active conformation. Therefore, we assume that phosphorylation would lead to stabilization of the activation segment in a conformation similar to that observed for PKA.Among the mitotic Nek kinases, there are no structures of Nek6 and Nek9, and there are only unphosphorylated structures of Nek2 and Nek7. However, these structures are sufficient to infer some details of their regulation by phosphorylation. Structures of Nek2 and Nek7 show disrupted hydrophobic spines and disordered activation segments (or activation segments in inactive conformations), and the Lys–Glu salt bridge is absent. These kinases need to undergo substantial changes in order to form an active conformation, and although we cannot at present say whether phosphorylation is sufficient to induce all of these changes, we know that phosphorylation is a key determinant of activity in all these kinases. Nek2 is activated upon autophosphorylation of Thr175 within the activation loop ,73, whicectively , and Nekectively ,38. Nek9ectively , but canectively ,104. Thea) [The phospho-regulation of Mps1 is complex and has several interesting features. Mps1 is capable of autophosphorylating multiple residues within its activation loop, and Thr675, Thr676 and Thr686 are all phosphorylated during mitosis ,105,106.figure 8a) –109. The8.a). This releases the activation segment from its autoinhibitory conformation, and remoulds the conformation of Cdk2 into a partially active form, in which the Lys–Glu salt bridge is formed, the hydrophobic spine is assembled and Thr160 is accessible for phosphorylation. One way of thinking about the function of the cyclin is that it completes the proper folding of the kinase domain, and that the Cdk on its own has an incomplete catalytic domain because it requires the cyclin for it to fold properly and have activity [Cdks are the best-known examples of protein kinases that are activated by the binding of a partner protein, which in this case are cyclins, and the details of the mechanism have been extensively reviewed ,10. Cycl4a) [e,f). When TPX2 is bound to Aurora-A, the phosphate group on Thr288 is buried, whereas without TPX2, this phosphate is accessible to phosphatases (e). This explains why dephosphorylation of Aurora-A by PP1 is less efficient in the presence of TPX2.The catalytic activity of Aurora kinases depends on the binding of a number of protein-binding partners and crystal structures of the Aurora-A/TPX2 and Aurora-B/inner centromere protein (INCENP) complexes have shed light on their respective activation mechanisms figures  and 9 8,86. TPX2figure 9a) . This inphatases e. This eThe structure of Aurora-B bound to INCENP, from the Musacchio group, shows a similar conformation of the activation segment to that observed in the Aurora-A/TPX2 complex . Residue−1, independent of the phosphorylation state of Aurora-A [More recently, the contribution of TPX2 binding and phosphorylation to Aurora-A activity have been thoroughly explored. TPX2 can stimulate the activity of unphosphorylated Aurora-A, and its energetic contribution to catalysis is 2.1–2.3 kcal molAurora-A . Our wor9.Bub1 and Haspin do not appear to be obviously regulated through any of the three common mechanisms discussed above. This is perhaps not surprising because they diverge hugely in their regulatory motifs . Althougb) [The primary sequence of Haspin is divergent from other protein kinases, including the catalytic and regulatory motifs . Crystalfigure 8b) ,116. Firb) .10.During mitosis, there is a peak in cellular phosphorylation, which is thought to effect a wholesale transformation in the activities of cellular proteins . These pProtein crystallography has revealed that active protein kinases adopt a specific conformation with respect to a key set of features, including the lysine–glutamate pair, the HRD and DFG motifs, the hydrophobic spine and a set of three basic residues that coordinate the activating phosphate group. These features are mostly conserved at the sequence level in mitotic protein kinases, although all mitotic kinases are divergent in at least one feature, which influences how they achieve an active conformation. Of the mitotic kinases, only Aurora-A, Plk1 and Haspin have been crystallized in fully active conformations, although the structures of Aurora-B/INCENP and Bub1 do exhibit most of the features of an active kinase. Perhaps most strikingly of all, structures of Haspin and Bub1 show how an active kinase conformation  can be achieved with highly divergent kinases despite lacking one of the key hydrophobic spine residues (Bub1) or several of the usual motifs (Haspin). In crystal structures of kinases in inactive conformations, these features are displaced from their active positions in a variety of ways, which have illuminated their mechanisms of regulation; for example, in the identification of a novel mechanism of autoinhibition in Nek7 and explaining why phosphorylation is insufficient for full activity in Aurora-A. Other structures raise the prospect of the existence of autoinhibited states  or highlight the requirement for further conformational changes to achieve full activity .10.1.bona fide phosphatase partners of other mitotic kinases, to understand the basis of their selectivity and regulation. Along these lines, it has recently been shown that PP6, not PP1, is the key Aurora-A inhibitory phosphatase and is capable of dephosphorylating the TPX2/Aurora-A complex [Mechanisms that prevent the untimely activation of kinases are crucial to ensure the fidelity of cell division. Autoinhibition is one such mechanism, as used in the regulation of kinases such as Cdk2 and BRAF. In this review, we have summarized the evidence for autoinhibitory conformations of mitotic kinases based on crystal structures of Aurora-A, Nek2 and Nek7. Further studies on these kinases are required to determine the contribution that these autoinhibitory states make to the physiological regulation of kinase activity. Future structural studies will reveal how these autoinhibitory states are reversed, for example through the interaction of Nek9 with Nek7. Reversal of activating phosphorylation by phosphatases is another mechanism deployed to prevent untimely kinase activation, and a clear example of this is the inhibition of Nek2 by PP1 . It is i complex . A clear10.2.Almost all mitotic kinases are regulated at some level by phosphorylation, and in many cases a site of activating phosphorylation has been identified on the activation loop . In Plk110.3.in vitro [Some mitotic kinases do not become fully activated until bound to a partner protein, triggering a conformational change, which may release an autoinhibitory state , reveal previously hidden additional phosphorylation sites (such as in cyclin activation of Cdks) or lock the protein in an active state (such as in TPX2 stimulation of phospho-Aurora-A activity). From a structural point of view, there are broad similarities between the ways that cyclins remodel the Cdk structure and TPX2/INCENP complement the catalytic domains of Aurora kinases. Not least of these is that the binding partners form crucial interactions with the α1/αC helix, and (at least in the case of cyclins and TPX2) with the activation segment. In this regard, they function similarly to the N- and C-terminal extensions of PKA . To datein vitro . It woul10.4.There are currently no structures of mitotic kinase catalytic domains bound to substrates, and although in some cases these interactions can be modelled with a degree of confidence, there are other examples for which an experimental structure would be very interesting . For exa10.5.Mitotic kinases are an important class of drug target, and understanding their regulatory mechanisms is key to effective new treatments for various hyperproliferative diseases, most notably cancer. Structural characterization of kinases facilitates rational drug design, enabling kinase active sites to be selectively targeted by tailor-made inhibitors. This has played a crucial role in the development of ATP-competitive inhibitors of Aurora kinases, Plk1, Mps1 and Nek2 ,122–124.In this review, we have described the remarkable progress in the resolution of mitotic kinase structures, and explained how these models have contributed to our understanding of their regulation and function. There are still many unanswered questions relating to the structural biology of mitotic kinases, and we have identified what we consider to be priorities for future work. Finally, we look forward to further developments in the field as the molecular details of mitotic signalling pathways are elaborated further, and this knowledge is translated into improvements in human health.11.R.B. is a Royal Society Research Fellow and acknowledges grant support from Cancer Research UK (C24461/A12772 and C24461/A13231). T.H. and S.Y. are supported by the University of Leicester. A.F. acknowledges grant support from The Wellcome Trust and the Association for International Cancer Research (AICR).


Mtl2p-GFP (green fluorescent protein) localized to the cell periphery and was necessary for survival under different types of cell wall stress. Wsc1p-GFP was concentrated in patches at the cell tips, it interacted with the Rho-GEF Rgf2p, and its overexpression activated cell wall biosynthesis. Our results are consistent with the notion that cell wall assembly is regulated by two different networks involving Rho1p. One includes signaling from Mtl2p through Rho1p to Pck1p, while the second one implicates signaling from Wsc1p and Rgf2p through Rho1p to activate glucan synthase (GS). Finally, signaling through the mitogen-activated protein kinase (MAPK) Pmk1p remained active in mtl2Δ and wsc1Δ disruptants exposed to cell wall stress, suggesting that the cell wall stress-sensing spectrum of Schizosaccharomyces pombe sensor-like proteins differs from that of Saccharomyces cerevisiae.Sensing stressful conditions that affect the cell wall reorganization is important for yeast survival. Here, we studied two proteins SpWsc1p and SpMtl2p with structural features indicative of plasma membrane-associated cell wall sensors. We found that Mtl2p and Wsc1p act by turning on the Rho1p GTPase. Each gene could be deleted individually without affecting viability, but the deletion of both was lethal and this phenotype was rescued by overexpression of the genes encoding either Rho1p or its GDP/GTP exchange factors (GEFs). In addition,  Schizosaccharomyces pombe cell wall mainly consists of an outer layer rich in galactomannoproteins (14%) and an inner layer of β-1,3-glucans (55%) and α-1,3-glucans (28%), all of which form a large complex - and α--glucans is regulated through the coordinated action of Rho1p and Rho2p, both of them GTPases of the Rho family  performs an essential function during the sporulation process and a secondary function redundant with Rgf1p during polarized growth -glucan synthesis through Pck2p. Rho2p binds to Pck2p and both are required for the activity and proper localization of the α--GS catalytic subunit (Mok1p/Ags1p)  proteins, the cell wall integrity and stress response component (WSC) family  and the Mid2p-Mtl1p pair  at an OD600 = 0.01.The genotypes of the wsc1::ura4+ disruption construct was obtained in a two-step process. The 5′ noncoding region of the wsc1+ open reading frame (ORF) (nucleotides [nt] −1010 to +34) was amplified by polymerase chain reaction (PCR), inserting the XhoI and HindIII sites (one at each end), and was ligated into the same sites of the SK-ura4+ vector. The 3′ flanking region of the wsc1+ ORF (nt +1027–2100) was amplified by PCR, inserting the BamHI and NotI sites as above, and was cloned into the same sites of pSK-ura+ with the 5′ end, to yield pCL1. wsc1+ gene disruption was accomplished using the 3.8-kb fragment from pCL1 cut with XhoI and NotI and transforming the YS64 haploid strain. Transformants were replica plated five times consecutively on YES medium to eliminate cells that had not integrated the construct. Integration was analyzed by PCR using the following oligonucleotides: M13 (5′-TTACCTAAGGCCACCAG-3′) in the ura4+ gene and 5′-comp-wsc1 (5′-CGTGGGTACTTCGACATG-3′) upstream from nucleotide +1153, and hence external to the disruption cassette. To make the wsc1::kan disruption construct (pRZ3), the uracil marker from plasmid pCL1 was excised and replaced by the kanamycin marker. wsc1::kan disruptants (GRG14 and GRG15) were obtained as above, tested for stability, and analyzed by PCR. To obtain the mtl2::his3+ disruption construct, the 5′ noncoding region of the mtl2+ ORF (nucleotides [nt] −1425 to −6) was obtained by PCR, inserting the ApaI and SalI sites (one at each end), and was ligated into the same sites of the pSK-his3 vector to yield pYS13. The 3′ flanking region of the wsc1+ ORF (nt +4433 to +5697) was obtained by PCR, inserting the PstI and NotI sites as above, and was cloned into the same sites of pYS13 to yield pYS2. mtl2+ gene disruption was accomplished using the 5.4-kb fragment from pYS2 cut with ApaI and NotI and transforming the YS64 strain. Correct integration was analyzed by PCR using the following oligonucleotides: M22 (5′-GTGTTCGCTAATTGCGA-3′) in the his3+ gene and Mid2-ext-5′ (5′-GTTGCTCTCATCCGTTG-3′) upstream from nucleotide −1290 and therefore external to the disruption cassette. For the mtl2::kan disruption construct (pSC13), the 5′ and the 3′ noncoding regions of mtl2+ were subcloned into the vector pKS-kan, proceeding as above.The wsc1 ORF), a NcoI-BamHI fragment containing the wsc1+ ORF was obtained by PCR and was subcloned into the same sites of pCL1 (described above). Based on the wsc1+ sequence, we designed oligonucleotides 400 bp apart and sequenced the entire ORF of four different clones. To tag Wsc1p at the C-terminus with GFP  and with the triple repeat of the influenza virus hemagglutinin epitope (HA)  was modified by site-directed mutagenesis. Site-directed mutagenesis was also used to create a NotI site immediately before the TAA stop codon of wsc1+ (pCL14). The GFP and HA epitopes were inserted in-frame at the NotI site of pCL14 to create pRZ11 and pRZ12, respectively. Strains with a genomic copy of wsc1+ tagged with GFP were obtained by one-step gene replacement. pRZ11 was modified to create a SmaI behind the wsc1+ ORF, allowing the insertion of the ura4+ marker at that site (pRZ21). A SpeI-BglII fragment of pRZ21 was used to transform a wild-type strain YS64, and correct integration of the fusion protein was analyzed by PCR. For wsc1+ overexpression, pCL10 (pAL-wsc1+) was modified by site-directed mutagenesis, introducing XhoI and SmaI sites flanking the wsc1+ ORF and thus creating pRZ15. The wsc1+ ORF from pRZ15 was cloned into the same sites of pREP3X and pREP81X, thus affording pRZ16 and pRZ17, respectively. To overexpress HA-tagged wsc1, a Nco1I-SmaI fragment containing the wsc1+ gene tagged with the HA epitope from plasmid pRZ12 (described above) was ligated into the NcoI-EcoRV sites of plasmid pJCR-L3x  was obtained by gap repair. Upstream and downstream flanking sequences from mtl2+ (from pYS2) were subcloned in pALKS. The plasmid was linearized with the 5′ and 3′ fragments at the ends and used to transform the wild-type haploid strain (YS64). The gap in the plasmid was repaired using the chromosomal sequences and the plasmids were recovered from the yeasts. Transformants were replica plated five times consecutively on YES medium and those able to lose the plasmid were selected. To tag Mtl2p at its C-terminus with the GFP, pSC2 was modified by site-directed mutagenesis. We created a NotI site immediately before the termination codon (pSC5). The GFP epitope was inserted in-frame at the NotI site of pSC5. pAL-mtl2+GFP (pSC7) fully complemented the mtl2Δ phenotypes. Strains with a genomic copy of mtl2+ tagged with GFP were obtained by one-step gene replacement. pSC9 was modified to create a SmaI at the 3′end of the mtl2+ ORF, allowing the insertion of a ura4+ marker at that site (pSC19). A 5.7-Kb SpeI-BstXI fragment from pSC19 was used to transform a YS64 wild-type strain.To make pCL10 (pALS. pombe cells were supplemented with U-[14C]-glucose (3 μCi/mL) and incubated for an additional 6 h at 28°C. To label cells overproducing wsc1+and mtl2+, cultures were induced for 14 h in the absence of thiamine before addition of U-[14C]-glucose (3 μCi/mL) and incubated for an additional 10 h. In both cases the radioactivity incorporated in the cell wall polysaccharides was measured as described . After incubation, the beads were washed several times and the bound proteins were analyzed by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) and Coomassie staining. The amount of GTP-bound Rho proteins was analyzed using 50 mL cultures of wild-type, wsc1Δ, and mtl2Δ cells containing HA-rho1+ expressed from its own promoter and performed as in Garcia et al. (The pGEX-C21RBD (rhotekin-binding domain) expression vector  growing at 28°C in YES medium and the appropriate stress treatment. Cell homogenates were prepared under native conditions employing chilled acid-washed glass beads and lysis buffer . The lysates were cleared by centrifugation at 13,000g for 10 min and Pmk-HA6H was purified with Ni2+-NTA-agarose beads . The purified proteins were loaded on 10% SDS-PAGE gels, transferred to an Immobilon-P membrane , and blotted to detect Pmk1-HA with 1:5000 diluted 12CA5 mAb as primary antibody  and with polyclonal rabbit anti-phospho-p42/44 antibodies (1:2500) . The immunoreactive bands were revealed with anti-mouse or anti-rabbit HRP secondary antibodies  and the enhanced chemiluminescence detection kit .Experiments designed to investigate Pmk1 activation under stress were performed using log-phase cell cultures  projections of z-series and were analyzed using deconvolution software from Applied Precision . Measurements were made from micrographs using the IMAGEJ  or METAMORPH  programs. For calcofluor white (Cfw) fluorescence, cells were harvested (1 mL), washed once, and resuspended in water with 20 μg/mL of Cfw at room temperature.Cell samples were visualized using an Olympus IX71 microscope equipped with a personal Delta Vision system and a Photometrics CoolSnap HQ2  monochrome camera. Stacks of seven S. pombe we performed a blast search  and two sequence orphans with certain similarity to ScMid2p, SpMtl1p (SPBC215.13) and SpMtl2p (SPAC11G7.01). However, we could not designate any of these proteins as SpMid2p because fission yeast Mid2p was already described as a component of the septin ring . The wsc1Δ and mtl2Δ mutants grew well under standard growth conditions at both 28 and 37°C and entered the stationary phase at the same time as the wild-type cultures. wsc1Δ and mtl2Δ cells did not exhibit any evident morphological changes as judged by light microscopy, but ∼8% of the cells in the wsc1Δ mutant and 15% of the cells in mtl2Δ were lysed , a lipopeptide antibiotic that inhibits β--glucan biosynthesis . The difference was mainly due to a decrease in the β–glucan content (17% in the wild type and 13% in the mtl2Δ) . However, microscopic observation of the germinated wsc1::his3+mtl2::ura+ null spores revealed that they were capable of polarity establishment and appeared to undergo a few cell divisions before growth stopped.We next looked at the effect of the mutations on mating and sporulation. The mating rate was not affected in P81nmt-mtl2 wsc1Δ (SC80), deleted for the wsc1+ gene and with the endogenous mtl2+ expressed under the control of the P81nmt promoter (P81nmt is a thiamine-regulatable and reduced-expression-rate promoter). Viable P81nmt-mtl2 wsc1Δ cells were obtained only when the 81nmt promoter was induced  could suppress the growth defect of the mtl2Δ mutants in the presence of Csp. As shown in Figure rho1+, rgf1+, and rgf2+ restored the growth of an mtl2Δ mutant in the presence of the antifungal agent, whereas overexpression of rgf3+ did not suppress the growth defect. Similarly, we asked whether upregulation of the protein kinase C homologues Pck1p and Pck2p, both targets of Rho1p in S. pombe, restored the growth defect of an mtl2Δ mutant in the presence of Csp. Both gene products – pck1+ and pck2+ – share overlapping roles in cell viability   (not shown), fully rescued the Csp hypersensitivity of mtl2Δ cells in rich medium.Because 1+) Fig. A or the wsc1Δ cells were defective in cell integrity signaling, we performed a suppression analysis of the wsc1Δ hypersensitive phenotype. Overexpression of Rho1p (pAL-rho1+) and Rgf1p (pAL-rgf1+) weakly suppressed the hypersensitivity of wsc1Δ cells to Csp  (not shown).To investigate whether Csp Fig. A. MoreovP81nmt-mtl2 wsc1Δ) unable to grow in the presence of thiamine (promoter off). The above findings strongly suggest that the essential cellular role of the putative sensors (Mtl2p and Wsc1p) is to activate Rho1p. Therefore, we analyzed the in vivo amount of GTP-bound Rho1p (activated-Rho1p) in cells with several different levels of Mtl2p and Wsc1p. In these experiments, GTP-bound Rho1p was obtained by precipitation with GST-C21RBD, the rhotekin-binding domain, in the extracts from the wild-type, the mtl2Δ and the wsc1Δ mutants, all of them containing HA-rho1+ expressed from its own promoter . Figure mtl2Δ and wsc1Δ mutants contained much less Rho1p-GTP than wild-type cells when the cultures were grown in the presence of 0.1 μg/mL of Csp for 16 h prior to harvesting  but it could not form colonies in the presence of thiamine (promoter of) (not shown). In the shut-off experiments, a high percentage of the cells lysed as long cells . However, regarding the Csp hypersensitivity phenotype, rgf2Δ was epistatic to wsc1Δ as the double-mutant wsc1Δrgf2Δ grew like the single rgf2Δ mutant in the presence of the drug  in the presence or absence of thiamine. In addition, we OP mtl2+ under the same conditions (pREP3X-mtl2+). When thiamine was eliminated to enhance wsc1+ or mlt2+ expression, the cells were unable to grow on plates (not shown). After 20 h of induction, Mtl2p overexpression produced long cells, probably due to cell separation defects, but otherwise their morphology was like that of the wild-type cells   . In these cells, the α-glucan fraction was twofold that found in the wild-type S. pombe cells, indicating a simultaneous increase in both α- and β-glucan polymers  Fig. C and thiers Fig. C. AdditiS. cerevisiae, activated sensors initiate the signaling cascade that eventually activates a MAPK (Slt2p) by local recruitment of Rom2p to sites near the cell surface where cell wall remodeling is required (Lessage and Bussey mtl2Δ or wsc1Δ mutants showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ions) characteristic of knockouts of components of the CWI pathway   Fig. B, sodiummtl2Δ cells could be overcome by upregulation or activation of the cell integrity pathway driven by Pmk1p. We found that the MAPK Pmk1p/Spm1p under the control of the strong and constitutively active alcohol dehydrogenase promoter . Expression of the Rho1p GEFs rgf1+ and rgf2+ and an effector of Rho1p, pck1+ rescued the growth defect in the presence of Csp, further supporting the notion that Mtl2p functions upstream Rho1p , pck2+, or the hyperactivation of Pmk1 MAPK by the expression of the constitutively active Pek1DD did not suppress sensitivity to Csp in the mtl2Δ cells. Third, Pmk1p signaling remained active in mtl2Δ and wsc1Δ disruptants exposed to cell wall stress and osmotic stress  and Rga8p (a Rho1p GAP). None of these genes is involved in Pmk1p activation.We found that neither a deletion of Mtl2p nor deletion in Wsc1p generates defects in the CWI pathway. First, knockout of Mtl2p and Wsc1p did not elicit the ess Fig. . Fourth,unk Fig.  have shoS. pombe sensor-like proteins differs from that of S.cerevisiae. This line of reasoning is supported by a report indicating that the central components of the osmotic, oxidative, and cell wall stress signaling pathways are relatively well conserved, whereas the sensors and transcriptional regulators of these modules have diverged significantly (Nikolaou et al. Our results therefore suggest that the cell wall stress-sensing spectrum of


In this review, the coherence of purine nucleoside-related pathways and MAPK activation in the endogenous neuroprotective regulation of the nervous system's development and neuroplasticity under hypoxic stress will be discussed.Even a short blockade of oxygen flow in brain may lead to the inhibition of oxidative phosphorylation and depletion of cellular ATP, which results in profound deficiencies in cellular function. Following ischemia, dying, injured, and hypoxic cells release soluble purine-nucleotide and -nucleoside pools. Growing evidence suggests that purine nucleosides might act as trophic factors in the CNS and PNS. In addition to equilibrative nucleoside transporters (ENTs) regulating purine nucleoside concentrations intra- and extracellularly, specific extracellular receptor subtypes for these compounds are expressed on neurons, glia, and endothelial cells, mediating stunningly diverse effects. Such effects range from induction of cell differentiation, apoptosis, mitogenesis, and morphogenetic changes, to stimulation of synthesis and/or release of cytokines and neurotrophic factors under both physiological and pathological conditions. Multiple signaling pathways regulate the critical balance between cell death and survival in hypoxia–ischemia. A convergent pathway for the regulation of multiple modalities involved in O In acute neurological conditions such as stroke severe injuries to the CNS occur  and stroFollowing hypoxia–ischemia, dying, injured, and hypoxic cells release soluble purine- nucleotide and -nucleoside pools , normallS-adenosyl homocysteine  and guanosine 5′-monophosphate (GMP) (Gs) or inhibit (Gi) adenylate cyclase, the enzyme that catalyzes the formation of cAMP, whereby A1R and A3R interact with Gi/ Go proteins, and A2A and A2B with Gs (2+- and mitogen-activated protein kinases (MAPKs), are also relevant (Adenosine receptors (AR) belong to the superfamily of G-protein-coupled receptors characterized by seven transmembrane helices . There aGs, except in striatum, where A2AR interacts with Golf, whereby both result in coupling to its canonical protein kinase A (PKA)-activating pathway -PKA pathway, the MAPK pathway, the stress-activated protein kinase  pathway, and the phosphatidylinositol 3-kinase-Akt pathway  cells  cells , which aensitive . Numerouensitive . Results2 receptor modulation of both voltage-sensitive potassium IK(V) and calcium ICa currents in PC12 cells (S-(4-nitrobenzyl)-6-thioinosine caused an increase in adenosine-mediated rescue of viability  pathway, serine–threonine kinases constitute a convergent pathway for the regulation of multiple modalities involved in O sensing  and regu sensing . Emergins module . Along ts module , that MAs module  and studs module . In cells module .MAPK was shown to positively modulate the hypoxia-inducible factor-1 alpha (HIF-1α) by phosphorylation control . Direct + channels and the activation of the phosphatidylinositol 3-kinase/Akt pathway (Next to the clear-cut effects of adenosine receptor-mediated activation of MAPK-HIF-1α, neuroprotection of hypoxic neuronal cells apparently does involve other pathways that deserve future attention. Amongst purine nucleosides guanosine attracted attention for its strong neurite-stimulating capacity ( pathway , made a  pathway  of poten pathway . Adenosi pathway .in vivo studies demonstrated inosine's ability to stimulate neurons to extend new projections to denervated areas in adult rats with unilateral cortical infarcts (In vitro studies confirmed the amazing neuroprotective capability of purine nucleosides in several neuronal hypoxia systems e.g PC12 cells and cerebellar granule neurons (2 sensing (Hypoxic–ischemic brain injury starts with the insult but extends into a recovery–reperfusion period . In para sensing . These r sensing . Amongst sensing . LikewisMany stroke patients fail clinical time windows for acute effective treatment, hence making approaches that promote repair and recovery essential for integrated stroke therapy . Thus, a


Combination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently BRAF inhibition has been proposed as pro-immunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and dendritic cell function. In this study we investigate the effects on isolated T-lymphocytes and monocyte-derived dendritic cells (moDCs) of a MEK and BRAF inhibitor combination currently being evaluated in a randomized controlled clinical trial.The effects of a BRAF inhibitor and a MEK inhibitor, alone and in combination were studied on isolated normal T-lymphocyte and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production and antigen-specific expansion, were assessed. moDC phenotype in response to lipo-polysaccharide stimulation was evaluated by flow-cytometry, as were effects on antigen crosspresentation.BRAF inhibition did not have an impact on T-lymphocytes or moDCs, whereas MEK inhibition alone or in combination with BRAF inhibition suppressed T-lymphocyte proliferation, cytokine production and antigen-specific expansion. Similarly, moDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. However no significant decrease in CD4+ or CD8+ T-lymphocyte viability was observed following kinase inhibition.MEK inhibition, alone or in combination with BRAF inhibition can suppress immune cell function and further studies in vivo will be required to evaluate the potential clinical impact of these findings.


Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the PM depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP6 phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth.Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues, internal stimuli can modify the activity or abundance of these proteins at the plasma membrane (PM). One such regulatory mechanism is protein phosphorylation by membrane-associated kinases, several of which are AGC kinases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the AGC kinases that most likely allow modulation of kinase activity by external stimuli . Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39  Cellular responses to external or internal stimuli include the fast modification of the already present machinery of proteins, which might eventually trigger the activation of novel transcriptional programs. A commonly used modification in cellular signaling is protein phosphorylation, which is achieved by the addition of a phosphate group to the side chain of an amino acid. In eukaryotes, protein kinases typically catalyze the transfer of a gamma-phosphoryl group from adenosine triphosphate (ATP) to a serine, threonine, or tyrosine in their substrate proteins. Other amino acids such as histidine, arginine, aspartate, and lysine are uncommon phosphorylation targets in eukaryotes, but often utilized in prokaryotes.Members of the protein kinase like superfamily have been found in genomes of archae, bacteria, and eukaryotes, highlighting the ancient origin of protein phosphorylation and its evolutionary conservation. With the arrival of multicellular eukaryotic organisms protein phosphorylation has been adopted to control a huge variety of cellular processes, and the involved kinases have evolved to fulfill new tasks, from triggering cell division to regulating membrane transport and cell polarity establishment. Eukaryotic protein kinases (ePKs) have been subdivided into 11 groups based on sequence similarity, evolutionary conservation, and known functions , cGMP-dependent kinases, and the diacylglycerol-activated/phospholipid-dependent kinase PKC. In the model plant Arabidopsis thaliana the AGC group consists of 39 members  in which groups AGC1 to 4 can be distinguished  are the only AGCVIII kinases that can be identified, indicating that these are the direct descendants of the first ancestral AGCVIII protein kinase , AGC2–2, UNICORN (UCN/AGC2–3), and UNICORN-LIKE (UCNL/AGC2–4). OXI1 and AGC2–2 have been shown to be involved in root growth  was found to interact with a kinesin-like protein  is highly variable between AGC kinases and extends over the N-lobe. While the N-tail of the PDK1s and AGC2 kinases is relatively short (25–50 amino acids) it is relatively large in several other AGC kinases , a T-loop , and a S/T]PxxGTx[D/E]Y) is necessary to activate the kinase  and the T-loop  association  stretches from the C-lobe around the kinase core and eventually folds back on to the N-lobe Figure B. Three The activity of protein kinases is regulated by posttranslational modification and by interacting proteins or macromolecules, both of which have an effect on their enzymatic activity and/or on their subcellular localization. The latter in turn determines the proximity to and thus the chance to phosphorylate their downstream targets. Most of these regulatory aspects involve the N- or C-terminal tail and the activation segment of the protein kinase. For the plant AGCVIII kinases, considerable research has been done on the BL receptors phot1 and phot2, and events leading to their activation are relatively well understood. Due to their role in regulating polar auxin transport, also PID, and two other AGC3 protein kinases (WAG1 and WAG2) have been analyzed in more detail. Compared to the phototropins, however, crystal structures and a detailed investigation of regulatory domains of the AGC3 protein kinases are missing. Below we will therefore summarize the current knowledge, and speculate on possible regulatory pathways with a focus on the AGC3 kinases and their role in PIN trafficking.As indicated in Section For PDK1, however, cis- and trans-autophosphorylation of its T-loop is not sufficient for activation, since it is initially auto-inhibited by its pleckstrin homology (PH) domain. This phosphoinositide binding domain bends over the catalytic cleft and blocks docking of protein substrates  that needs to be phosphorylated before interaction with PDK1 can occur. In the mammalian AGC kinases PKCζ and PKC related kinase 2 (PRK2) an aspartic acid and glutamic acid residue are respectively present at the serine/threonine position, and these residues are required for binding of and activation by PDK1, probably because they mimic the negative charge of a phosphorylated serine/threonine  have been identified. Based on the presence of a PIF domain in several plant AGC kinases, as well as on protein–protein interaction studies and in vitro activity assays, Arabidopsis PDK1 potentially is involved in T-loop phosphorylation and activation of at least 16 AGC kinases . Osmotic stress leads to removal of RAPTOR and the subsequent loss of PIF domain phosphorylation by TOR, which inhibits PDK1 dependent activation of S6K1  phosphorylation motif of the form FxxFE. coli expressed PKA has been reported to auto-phosphorylate in vitro . Nuclear localization of BIF2 might be mediated through its interaction with BA1 or through a yet unidentified nuclear localization signal  has been identified. Like PID, a YFP:BIF2 fusion localizes to the PM and phosphorylates the HL of ZmPIN1a The actual protein domains involved in PM association of phot1/2 and PID are not known. A transmembrane domain is clearly absent in these proteins, implying that PM association is achieved by interaction with other PM-bound proteins or through binding to lipid components of the PM. Binding of PID to several phosphoinositides and phosphatidic acid (PA) in a lipid-overlay assay suggested that the latter is true. A fusion of GFP with the insertion domain of PID localizes to the PM in yeast cells, demonstrating that this domain is sufficient for PM localization. This is further substantiated by the redirection of AGC1–7 from the cytoplasm to the PM in tobacco cells after exchange of its insertion domain for the PID insertion domain  and completely inhibited by another  that have their own calmodulin (CaM) domain with four calcium binding pockets (EF-hands) to sense the calcium concentration. Binding of Ca2+ has been reported for the conventional animal PKC isoforms. Calcium binding to the C2 domain enhances PM association and subsequent activation of PKCs  and the CaM-like protein TOUCH3 (TCH3). Binding of these proteins to PID is enhanced by calcium. However, while PBP1 interacts weakly with PID in the absence of Ca2+, binding of TCH3 is strictly Ca2+-dependent. Importantly, autophosphorylation of PID is enhanced by addition of PBP1 in the kinase assay, while it is repressed by TCH3   could be detected after 30 s of BL irradiation. A maximum of PKS4L was detectable after 10 min of irradiation, after which PKS4L levels decreased and finally were no longer detectable after 4 h of irradiation. PKS4L was detectable in in vitro and in yeast cells. In HeLa cells co-transfected with ABCB19 and phot1 auxin efflux is abolished after BL treatment, indicating that phosphorylation of ABCB19 by phot1 inhibits the auxin transporter. Further immuno-precipitation assays showed that the interaction between TWD1 and ABCB19 is lost after BL treatment of wildtype seedlings, but can be detected in equally treated phot1 seedlings. This indicates that phot1-dependent phosphorylation of ABCB19 disrupts the positive modulation of ABCB19 by TWD1 at the light side of the hypocotyl  has been suggested to act as a positive modulator of its activity  PIN localization, whereas loss-of-phosphorylation was shown to induce a PIN polarity shift toward the basal (rootward) cell membrane  from a symmetric distribution to an asymmetric localization at the inner lateral side of hypocotyl endodermis cells. Genetic evidence was provided that at least the PID kinase is involved in this response. The observed phot1-dependent down regulation of PID expression was proposed to lead to PIN3 loss-of-phosphorylation and thus to GNOM dependent trafficking of PIN3 proteins to the inner lateral side  that acts redundantly to TWD1  with other eukaryotes, but lack the typical PKA and PKC subfamilies that are involved in regulating cell growth, -division, and -polarity in animals and yeast. Instead, plants seem to have adopted the AGCVIII kinase subfamily to perform similar functions. The first AGCVIII kinase appeared in unicellular algae as BL-sensing photoreceptor (phot2), whose signaling protects the cells against high fluency rate BL. During the evolution of multicellular plant life and the move from water to land, the AGCVIII kinases diversified by losing the photo-sensing domain, and were recruited for other functions such as control of auxin transport  and pathogen responses (OXI1)  the necessity of T-loop phosphorylation for full activation, and (ii) the potential involvement of PDK1 in this activation, including its interaction via the C-terminal PIF domain. It remains to be determined, however, how plant AGC kinases are activated. With PDK1 a general upstream activator of AGC kinases in plants might be present, but at least for the phototropin kinase domains and several other AGC kinases  auto-activation has been reported. For PID, activation was stronger in the presence of PDK1 . It will be interesting to see which intrinsic signals instruct their subcellular localization, and whether this is modulated in response to distinct stimuli. Further dissection of the AGC kinase subdomains guided by the structural knowledge presented in this review will help to understand the dynamics of intracellular localization of plant AGC kinases in relation to the phosphorylation state of their targets and the processes that they regulate.Next to activation of the T-loop we have highlighted the potential regulatory domains in the insertion domain of the activation segment and in the N- and C-tail that extend from the kinase core of plant AGC kinases. As in animal AGC kinases, the relatively large N- and C-tails of plant AGC kinases suggest that protein–protein interaction domains may be found here. The insertion domain that is typical for the plant specific AGCVIII kinases was found to be essential for proper subcellular localization of Lotus japonicus PKL01, an NDR kinase (AGCVII) homolog, describes this AGC kinases as being able to phosphorylate tyrosine residues next to its conventional serine/threonine kinase activity (Katayama et al., Arabidopsis has not been determined yet. It will be interesting to see if this expanded function is present in Arabidopsis, and which structural changes might enable the dual-specificity of the NDR kinases.The identification of novel AGC kinases can be surprising as a recent report on Arabidopsis AGC kinases have been characterized in other plant species (see Table Medicago truncatula. In Arabidopsis this kinase has been shown to regulate root hair elongation (Oyama et al., Medicago a role in the formation of nodules has been described (Pislariu and Dickstein, Arabidopsis, or due to a changed role of IRE in Medicago remains unknown, and mis-expression experiments with the two proteins as well as identification of substrates in the respective plants will be needed.Furthermore, homologs of In conclusion, plant AGC kinases are an interesting group of kinases that perform functions conserved in all eukaryotes, but also plant-specific functions. Especially the plant-specific AGCVIII kinases have several interesting features, such as their dynamic subcellular localization, and we are confident that further dissection of their structure and function will lead us to new paradigms in signaling and cell polarity regulation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga. Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets.The methane-emitting cold seeps of Hikurangi margin  are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic  Ampharetidae, Polychaeta)  remained below 3 µM down to 16 cmbsf (centimeter below sea floor) and sulfate [SO42-] was nearly constant at 28 mM. Methane oxidation (MOx) was not detected and sulfate reduction (SR) was present at very low rates (4] reached up to 600 µM and [SO42-] decreased from 28 mM to 0 mM within the upper 20 cmbsf. The sulfide concentration at site 124 was low in the upper sediment horizon (0-7 cm), but increased strongly below. Although sulfide data were not available for site 309, this trend was observed at this and other ampharetid sites (-2 yr-1 (mean ± standard error) at site 124 and 1.8 ± 0.2 mol m-2 yr-1 at site 309. In the sulfidic layers integrated MOx rates were higher with 4.3 ± 1.2 mol m-2 yr-1 (site 124) and 6.1 ± 1.9 mol m-2 yr-1 (site 309). Integrated SR rates were 5.0 ± 1.9 mol m-2 yr-1 (site 124) and 2.9 ± 0.8 mol m-2 yr-1 (site 309). The average annual methane oxidation rate found in the oxic layer of all ampharetid sites was around 0.7 ± 0.2 mol m-2 yr-1, as compared to 3.7 ± 1.1 mol m-2 yr-1 in the anoxic zone.Biogeochemical measurements were conducted at all seep sites with a focus on the ampharetid habitats 124 and 309, the SOB habitat 315 and the frenulate habitat 45. We distinguished between oxic/suboxic and sulfidic sediment layers based on sulfide concentration or appearance of the sediment cores. At the frenulate site 45 methane concentrations [CHow rates . At the id sites , indicatid sites , which wid sites . Within -2 yr-1 within the upper 10 cmbsf and 9.1 ± 2.7 mol m-2 yr-1 between 10 and 20 cmbsf. Integrated SR rates were 21.4 ± 2.9 mol m-2 yr-1 and 3.7 ± 0.9 mol m-2 yr-1, respectively. At the reference site we did not detect methane, sulfide, AOM or SR  resulted in a total of 305 partial and 21 full-length, high quality sequences. We found the highest archaeal richness at the reference site with 27 observed and 48 estimated operational taxonomic units (OTUs) using Chao1 at a 97% similarity cut-off. The lowest archaeal richness was present at the SOB site (14 observed and 25 estimated OTUs). At the frenulate and ampharetid sites 16 of 28 and 18 of 46 estimated OTUs were retrieved, respectively.Thaumarchaeota and Crenarchaeota of Marine Benthic Group B, whereas sequences of methane-oxidizing groups were absent  yielded a total of 355 bacterial 16S rRNA partial and 78 full-length sequences. These were distributed widely among the Bacteria . The higAlphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria, Planctomycetes and Acidobacteria, which were present in all sediments. Deltaproteobacteria was the dominant phylum in all libraries and the majority of the clones was assigned to sulfate reducers   . MMG1 anpmoA genes encoding for subunit A of the particulate methane monooxygenase. This enzyme plays a central role in aerobic methanotrophy as it activates methane by the addition of oxygen. Analysis of the top 10 cm of sediment at sites 45, 309 and 315 yielded 236 sequences and 24 OTUs. The Chao1 estimated richness was 29 OTUs. The OTUs were calculated using a cut-off of 93% amino acid sequence similarity, which corresponds to a 97% 16S rRNA similarity [pmoA composition at the frenulate site 45 (20 OTUs). Many of the OTUs were related to endosymbionts of Lamellibrachia sp. and did not occur at the other seeps. The pmoA diversity at the SOB site 315 was also quite high (8 OTUs), but the OTUs mostly differed from the ones present at the frenulate site. Interestingly, at the ampharetid site 309 we discovered only 2 different OTUs. Both OTUs were closely related to methanotrophic endosymbionts. The pmoA sequences formed five very distinct monophyletic clusters , for one SOB site (315) and one frenulate site (45). The percentages of subgroups are given relative to the total number of cells, including aggregates. Total single cell numbers  do not c9 cells ml-1 sediment , of which Methylococcales (probe MTMC-701 and competitors) dominated the surface sediment at both sites (12-15%). However, as many of the observed Methylococcales aggregated in clusters of irregular shape, their total abundance was certainly underestimated. Methylococcales aggregates were between 5 and 40 µm in diameter  also visualized a few up to 30 µm long filaments with square-shaped cells  and oval or round cocci (up to 2µm) showing a high morphological diversity. MMG1 (probe MetI-444) comprised cells that were stained by a probe specific for Bathymodiolus spp. endosymbionts  and potential sulfur-oxidizing Gammaproteobacteria  that occurred in the oxic surface layer. Single cells of sulfate-reducing Desulfosarcina/Desulfococcus (probe DSS658) occurred throughout the sediment (1-14%) and peaked in the anoxic layer.The highest total cell numbers at the ampharetid sites were found in the oxic top centimeter. Most cells were identified as diameter . The lardiameter . Small adiameter . Many aged cells . Single Archaea were almost absent in the upper oxic centimeters, but their cell numbers increased with depth. At site 124, AOM consortia of ANME-2a (probe ANME2a-647) and SEEP-SRB1a (probe SEEP1a-473) were largest and most abundant between 7 and 13 cmbsf, peaking with 4.2 × 107 consortia ml-1 sediment  with an average of 2.9 ± 0.3 µm (about 140 cells). Hence, the total number of AOM-mediating archaeal and bacterial cells at this site may have been as high as 5.9 × 109 ml-1. We found many small AOM aggregates, lesser medium sized ones and only few large aggregates . Similar to site 309 we again found about 1% MMG1 and 1% MMG3, while MMG2 was almost absent. Cell numbers of aerobic methylotrophs decreased within the first 3 cmbsf and were not detected in the deeper sediment. The bacterial community of the top layer included organisms involved in sulfur cycling, such as potential sulfur-oxidizing Gammaproteobacteria (up to 3%) and Epsilonproteobacteria (up to 4%). Single cells of the DSS group occurred in all depths (up to 12%).The sulfidic top centimeter of sediment at the SOB site also harbored the largest number of cells with ANME-2 archaea were omnipresent and mostly associated with DSS. Relative abundance of ANME-2  and DSS (30%) peaked between 2 and 8 cmbsf. Single cells of ANME-2 (up to 4%) and DSS (up to 15%) were also abundant. We found the highest aggregate numbers at 2 cmbsf, whereas the largest aggregates, with diameters of >16 µm, occurred at 6 cmbsf. In the uppermost layers, ANME-2a/DSS aggregates dominated, whereas in the deeper sediment, ANME-2a and ANME-2c were equally abundant. Neither ANME-1 nor ANME-3 archaea were detected. Methanogenic archaea (probe MS1414) made up 2-3% of all cells in the horizon between 2 to 9 cm, but were absent above and below.To get a broader perspective of the microbial communities on Hikurangi margin we assessed the variation of microbial diversity between different seep sediments by ARISA. In total 385 different OTUs  were found . Each 5 Grouping the dataset according to the habitats, i.e. the dominant seep-associated fauna, showed the differences between all seep habitats and the reference site . 169 OTUAlthough each seep ecosystem contained a comparable number of OTUs (225 ± 30), their OTU composition differed significantly . The hig-2 d-1 (mean = 84 mmol m-2 d-1) [Many of the cold seeps at Hikurangi margin investigated during expedition SO-191 leg 2 and 3 were densely populated by heterotrophic ampharetid polychaetes ,8. Thesem-2 d-1) ,10,11,1213C-depleted carbon isotopic signature of their tissue clearly revealed a methane-based nutrition [In turn, the heterotrophic polychaetes seemed to feed mainly on the methanotrophs, since the utrition . Infaunautrition . Thus, iMethylococcales and MMG1 were reported from oxygen-depleted sediment [pmoA sequences related to the ones we observed in ampharetid habitats were found in oxygen minimum zones of the Eastern Pacific Ocean [Methylococcales while MMG3 is closely related to the deep-branching Methylophaga, which are non-methane utilizing methylotrophs. MMG1 are so closely related to endosymbionts of Bathymodiolus spp. that we visualized the cells in the sediment with a Bathymodiolus endosymbiont specific probe. MMG1-3 occurred also at the SOB habitat, whereas MMG3 was missing at the frenulate habitat.We confirmed a large community of aerobic methanotrophic bacteria in the uppermost oxic-suboxic sediment layers at ampharetid habitats, whereas anaerobic methane-oxidizing archaea dominated the deeper anoxic and sulfidic layers. The aerobic methanotrophs clustered into three monophyletic clades, which we named Marine Methylotrophic Group (MMG) 1-3, because they contain sequences retrieved from marine methane-rich ecosystems or methanotrophic endosymbionts. Sequences of sediment  and pmoAic Ocean . Thus, mic Ocean , and weric Ocean . MMG1 anpmoA gene, revealed five monophyletic clusters that exclusively contained sequences originating from marine methane-rich ecosystems. The high diversity of pmoA sequences present at the SOB and frenulate habitats supported the results of the 16S rRNA gene analysis. We found organisms related to benthic and pelagic methanotrophs at both ecosystems and a high number of OTUs related to siboglinid endosymbionts at the frenulate habitat. However, the ampharetid habitat had a much lower pmoA diversity as was expected from the 16S rRNA analysis. All pmoA sequences clustered into two OTUs (at a 93% similarity cut-off) forming two distinct clades. One clade contained pmoA sequences related to Bathymodiolus spp. endosymbionts, while the other contained sequences related to Rimicaris sp. epibionts. The OTUs likely belonged to MMG1 or MMG2, since members of the MMG3 might lack a pmoA gene like their close relatives of the genus Methylophaga [pmoA diversity might be due to a PCR bias caused by a clear dominance of these two OTUs in the sediment. MMG2 strongly dominated the methylotrophic community and contributed by far most of the biomass in the form of large aggregates. Thus, only two taxa seem to be responsible for most of the aerobic methanotrophy at ampharetid habitats despite the high diversity of methylotrophic communities found in Hikurangi ecosystems. This low diversity could be the result of a natural enrichment of aggregate-forming organisms of MMG2 caused by the polychaete activity. The existence of a specific niche at ampharetid habitats is supported by the absence of alphaproteobacterial type-II methanotrophs. We did not find them in 16S rRNA and pmoA gene libraries from the investigated cold seeps, although they are abundant in nearby methane-bearing sediments [The analysis of aerobic methanotrophs on a functional level, using the lophaga . The lowediments .The anaerobic microbial communities at ampharetid habitats were confined to sulfidic sediment layers below the oxygenated zone. They were dominated by ANME-2/DSS consortia known to mediate AOM . AlthougHikurangi margin cold seeps are far away from all studied seep areas and only few deep-sea chemosynthetic ecosystems are known from the Southern Hemisphere ,61,62.PARISA data revealed a comparable richness of abundant bacterial types at each site and in the four depth layers investigated. However, β-diversity was considerably different at all spatial scales from a few centimeters to hundreds of kilometers. For example, microbial communities in two depth layers of the same ecosystem frequently shared as little or less OTUs as two seep ecosystems from different geographical regions. The observed increasing dissimilarity of the community with increasing sediment depth was already described in previous studies ,64. ThisHikurangi margin harbors cold seep ecosystems with unique features concerning seep-associated fauna and methanotrophic communities. Bioirrigation by the ampharetid polychaetes maintains a habitat suitable for aerobic methylotrophic bacteria in the top sediment, contributing to about 25% of total methane consumption, while anaerobic methanotrophic archaea dominate the deeper anoxic layers. Dense polychaete communities such as the ones formed by ampharetids investigated here are present also at other seepage sites ,66,67. AFigure S1Biogeochemistry of additional Hikurangi ecosystems.(PDF)Click here for additional data file.Figure S2Phylogeny of archaeal 16S rRNA.(PDF)Click here for additional data file.Figure S3Phylogeny of deltaproteobacterial 16S rRNA.(PDF)Click here for additional data file.Figure S4pmoA protein.Phylogeny of (PDF)Click here for additional data file.Figure S5Bathymodiolus spp. Endosymbionts.Micrographs of organisms related to (PDF)Click here for additional data file.Figure S6Abundance and size distribution of AOM aggregates.(PDF)Click here for additional data file.Figure S7Number of operational taxonomic units at each site.(PDF)Click here for additional data file.Figure S8Partitioning of operational taxonomic units.(PDF)Click here for additional data file.Figure S9Shared operational taxonomic units between sites.(PDF)Click here for additional data file.Figure S10Redundancy analysis of ARISA data and environmental parameters.(PDF)Click here for additional data file.Movie S13D animated methylococci aggregate.The methylococci aggregate originates from the oxic layer at ampharetid site 124. The cells are hybridized with the Methylococcales specific probe  and nucleic acid is stained with DAPI (blue). The aggregate consists of large oval cells that are loosely packed. For details concerning the animation see Materials and Methods S1.(MPG)Click here for additional data file.Movie S23D animated AOM aggregate.The AOM aggregate was found in the anoxic layer of ampharetid site 124. The diameter was 12 µm, total cell volume of the aggregate (excluding intercellular space), based on the DAPI signal, was 76 µm3, the volume of the archaeal core (ANME-2a-647 - red) was 20 µm3 and the bacterial shell (SEEP1a-473 - green) was 25 µm3. For details concerning animation and volume calculation see Supporting Material and Methods.(AVI)Click here for additional data file.Table S1Total cell counts and relative abundance of methanotrophs at Hikurangi margin, Hydrate Ridge, Black Sea and Håkon Mosby mud volcano seep sites.(PDF)Click here for additional data file.Materials and Methods S1(PDF)Click here for additional data file.


Borders are important as they demarcate developing tissue into distinct functional units. A key challenge is the discovery of mechanisms that can convert morphogen gradients into tissue borders. While mechanisms that produce ultrasensitive cellular responses provide a solution, how extracellular morphogens drive such mechanisms remains poorly understood. Here, we show how Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) pathways interact to generate ultrasensitivity and borders in the dorsal telencephalon. BMP and FGF signaling manipulations in explants produced border defects suggestive of cross inhibition within single cells, which was confirmed in dissociated cultures. Using mathematical modeling, we designed experiments that ruled out alternative cross inhibition mechanisms and identified a cross-inhibitory positive feedback (CIPF) mechanism, or “toggle switch”, which acts upstream of transcriptional targets in dorsal telencephalic cells. CIPF explained several cellular phenomena important for border formation such as threshold tuning, ultrasensitivity, and hysteresis. CIPF explicitly links graded morphogen signaling in the telencephalon to switch-like cellular responses and has the ability to form multiple borders and scale pattern to size. These benefits may apply to other developmental systems. During development, morphogen gradients play a crucial role in transforming a uniform field of cells into regions with distinct cell identities (marked by the expression of specific genes). Finding mechanisms that convert morphogen gradients into sharp borders of gene expression, however, remains a challenge. Cellular ultrasensitivity mechanisms that convert a linear stimulus into an on-off target response offer a good solution for making such borders. In this paper, we show how a cross-inhibitory positive feedback or toggle switch mechanism driven by two extracellular morphogens – BMP and FGF - produces ultrasensitivity in forebrain cells. Experiments with cells and explanted brain tissue reveal that BMPs and FGFs cross inhibit each other's signaling pathway. Such cross inhibition could occur through four possible mechanisms. By an iterative combination of modeling and experiment, we show the toggle switch to be the mechanism underlying cross inhibition, the ultrasensitive expression of multiple genes, and hysteresis in forebrain cells. As the toggle switch explicitly links extracellular morphogens to cellular ultrasensitivity, it provides a mechanism for making multiple sharp borders that can also scale with tissue size – an important issue in pattern formation. This might explain the abundance of BMP-FGF cross inhibition during development. The formation of borders between compartments and body parts is crucial for embryonic development Extensive studies in many systems Msx1 and Ttr being expressed in the CPE Msx1 is a high-threshold BMP target gene in many patterning systems Ttr is induced specifically in the CPE at the onset of its definitive differentiation  and is stably expressed thereafter One system patterned by interacting morphogens is the dorsal telencephalon Msx1 is restricted to the midline, BMP4 can induce Msx1 expression in dissociated cortical precursor cells (CPCs) in an ultrasensitive fashion in vivo and in vitro, Msx1 ultrasensitivity contrasts with graded changes in nuclear phospho-Smad1, 5, or 8 (pSmad) levels , within the same cells. This implies that Msx1 ultrasensitivity occurs downstream of pSmad activation. The mechanism underlying this ultrasensitivity, however, remains unknown.Although Dorsal telencephalic cells responsive to BMP also respond to other morphogens, such as FGFs (most notably FGF8) produced in the adjacent rostral midline (RM) and cortex We investigated the influence of FGFs on DM BMP target genes, and found that ultrasensitivity requires cell-intrinsic interactions between the BMP and FGF pathways. Using explants and dissociated cell cultures, we showed that the BMP and FGF pathways mutually inhibit at the single cell level; Epidermal Growth Factor (EGF) acts similarly to FGF. Next, we used modeling to identify experiments that distinguish among different models of cross-inhibition. These experiments identified a cross-inhibitory positive feedback (CIPF) mechanism, or “toggle-switch”, between the BMP and FGF signaling pathways as the basis for ultrasensitivity. We further show how this mechanism is capable of generating multiple sharp borders simultaneously, among other potential advantages.Ttr and Msx1 regulation in the dorsal telencephalon, we used two previously-characterized in vitro systems: a dorsal forebrain explant system and dissociated cultures Ttr expression towards the RM , with the Ttr-expressing cells often bending inward towards the ventricle .To experimentally study entricle ; these fexplants . SimilarMsx1 was ectopically induced in the cortex  Ttr levels peaked at a BMP4 concentration of 16 ng/ml . These explants displayed rostral Ttr expansion reminiscent of that seen in BMP4-treated explants  Ttr  in midlitr  at low BMP4 concentrations (1.5 ng/ml) with and without FGF2 (10 ng/ml), 2) at mid-level BMP4 concentrations (16 ng/ml) with and without FGF2, and 3) at mid-level BMP4 concentrations and FGF2 with and without PD173074 (100 nM). As expected, Msx1/2 positivity and expression levels in CPCs were higher at 16 ng/ml than at 1.5 ng/ml BMP4 . The treatment resulted in dose-dependent decreases in (mRNA) Msx1 levels, while (mRNA) Spry1 levels increased  ne cells . When BMll types . Thus, Bncreased . These rSpry1 expression. Taken together, the experimental data indicate that BMP and FGF signaling inhibit each other's target responses, and that this mutual or cross inhibition is intrinsic to both midline and cortical cells.As an additional test for BMP4 inhibition of the FGF pathway, we examined how BMP4 affected FGF-stimulated cell proliferation Ttr and Msx1 and BMP4 directly inhibits Spry1 (I and BI), and target responses (FT and BT). Inhibitory links between the pathways were then introduced, resulting in 81 possible configurations  simple target inhibition (STI), 2) simple upstream inhibition (SUI), 3) coherent feedforward (CFF), and 4) cross-inhibitory positive feedback (CIPF). Representative models that captured the basic response dynamics of each class are shown in T), interaction and nodes that have no influence on BT (e.g. FT) are omitted from these depictions. Later, we will argue that the particular selections of inhibitory connections in the representative models are likely to be well justified  in the presence or absence of a fixed amount of FGF. Under these circumstances, three features of the BT response are potentially informative: maximal levels, EC50 values, and sensitivity. The sensitivity could be either linear (hyperbolic) or ultrasensitive  to varying degrees, as quantified by its apparent Hill coefficient, or nH. Changes in these response features were evaluated over a wide range of parameter space and across different “contexts” in which different links within the models were made nonlinear to different degrees  Msx1 levels, while the EGF receptor inhibitor PD153035 50 = 25 pM) produced dose-dependent Msx1 upregulation  maximal (mRNA) Msx1 levels at high BMP4 concentrations were unchanged, 3) the EC50 increased (1.5 to 8.3 ng/ml), and 4) marked ultrasensitivity emerged. These characteristics precisely matched the CFF (with nonlinear inhibitory links) and CIPF models  correlated strongly  or BMP4 at different concentrations (4–64 ng/ml) was reapplied before harvesting CPCs two days later  to induce the ys later . In CPCsMsx1 did indeed return to low baseline levels  or high BMP4 (Msx1-on), (mRNA) Msx1 levels were higher in the Msx1-on CPCs regardless of the BMP4 concentration that was reapplied after washout  was significantly lower than that of Msx1 (32 ng/ml BMP4) in the same cells  embryos which express nuclear lacZ in the Msx1 domain Msx2 expression domain extends ∼100 µm farther than that of Msx1  would be ∼270 µm. This value agrees well with the dorsoventral pSmad gradient in the E10.5 dorsal telencephalon Msx1 and Msx2 EC50 values in vitro correlate well with their expression borders in vivo.For morphogen thresholds ∼1.5 fold apart  by BMP and FGF or differential loop inhibition between the intermediates - or downstream of the CIPF loop. Our simulations show that the first category produced BT responses with separate EC50 values  within a gradient  and its apparent irreversibility Ttr is more complex than Msx genes, which are direct and immediate BMP targets. The differences between Ttr and Msx1 in the BMP4- and FGFR inhibitor-treated explants also raise the possibility that Ttr and Msx1 are regulated by different FGFs (e.g. FGF8 and FGF2). Although changes in cell composition after two days in culture may contribute to observed findings, previous two-day cultures on more naïve E10.5 CPCs yielded similar ultrasensitive Msx1 responses The complete elimination of  and EGF , 5 indicsitivity . Consiste cortex . This imIn vivo, pSmad1/5/8 immunoreactivity declines gradually and smoothly with distance from the DM in vitro, in the presence of FGF2 and EGF, nuclear pSmad levels exhibit a graded relationship to BMP4 dose while Msx1 and Msx2 inductions are ultrasensitive  or a single extracellular morphogen and a transcriptional network  Cross-inhibition (or cross-repression) is, of course, not new in developmental biology  balance each other. The effects of BMP4 or FGFR inhibitors on forebrain explants  of the University of California, Irvine. All experiments were conducted in accordance with protocol # 2001–1024 approved by the Institutional Biosafety Committee (IBC) of the University of California, Irvine. All surgeries were performed on euthanized animals with all efforts made to minimize suffering. Animals were euthanized with carbon dioxide from compressed gas cannisters, with secondary physical method of cervical dislocation to ensure euthanasia.Msx1-nLacZ mice Noon of vaginal plug date was day 0.5; developmental stages were confirmed by crown-rump measurement. CD1 mice  were used for wild-type studies. 5) and run in duplicate or triplicate for 40 cycles; cyclophilin A (CYPA) and 18S reference primers were included in runs , except for explant studies (CYPA only). Mean, SEM, SD, and p values  were calculated from cycle threshold (dCt) values (Ctgene of interest – Ctreference) and plotted as normalized ddCt values (upregulation is positive and downregulation is negative). In RNA preps, cDNA syntheses, PCR quality controls, experimental runs, and statistical methods were performed as described previously Ttr, Msx1, Spry1) to maintain specificity for midline cells. After adhering overnight, human recombinant BMP4 , FGF8, FGF2, EGF , heparin , PD153035, SU5402 , PD173074 , and/or LDN193189  were added at indicated concentrations. All RNA purifications  were done 48 hrs after initial BMP4 treatment except for the following experiments: 1) midline cells treated with FGF8 . Cells were plated in 96 well plates at matched densities from 25,000–50,000 cells/well. 24 hrs post-plating, BMP4, FGF2, FGF8, and/or EGF at the indicated concentrations was added to media . WST1 waImmunohistochemistry was performed as described 50 values were obtained by fitting the BT response to a Hill equation using the Mathematica FindFit function. The maximal values were measured by computing the BT response at an extremely high BMP dose to account for asymptotic behavior. For 50, and nH) were assessed for all four models .For 50 values were obtained using the curve fitting function of Deltagraph 6.0. The length scale of the pSmad gradient in the dorsal telencephalon was based on previous data x the position in µm, λ (lambda) the length scale, A the highest concentration without b, the background.In Each of the models was defined by a set of ordinary differential equations. The form of these equations and their rationale is similar to previous modeling approaches The four models shown in I inhibits BT:Simple Target Inhibition (STI) – FI inhibits BI:Simple Upstream Inhibition (SUI) – FI inhibits BT, BI inhibits FI:Coherent Feedforward (CFF) – FI inhibits BI, BI inhibits FI:Cross Inhibitory Positive Feedback (CIPF) – FExplanation of parameters:1, kc3, kc5kc – maximum rates of production (Moles/sec)tb, Ktf, KtbtK – half saturation constants of activation (M)2, kc4kc – half saturation constants of inhibition (1/M)BI, KFI, KBTK – degradation rates (1/s)n1, n2, n3 – Hill coefficientsTo reduce the number of parameters and simplify analysis, these equations were non-dimensionalized  Forebrain schematic  showing the DM region where BMP4 is produced (orange), which forms between the two cortical hemispheres (green). FGF2 is produced in the cortex, whereas FGF8 is produced in the rostral midline (blue). (B) RT-qPCR of the rostral and dorsal halves of explants. DM-specific genes are upregulated in their endogenous  and ectopic domains  in BMP4-treated explants compared to BSA-treated controls  Sections of whole-mount explants shown in Ttr ectopically in a 1–2 cell-thick layer that bends toward the ventricle, thus resembling endogenous CPE. (D) In situ hybridization (ISH), en face images of whole mount explants. Compared to control (BSA-soaked) beads , FGF8-soaked beads suppress endogenous Ttr expression (n = 8/12 explants). (E) RT-qPCR and WST1 assays, E12.5 midline cells. The FGFR inhibitors PD173704 (left) and SU5402 (right) downregulate the FGF-target gene Spry1. 16 and 32 ng/ml FGF8 (no BMP4 present) increases midline cell numbers, while 8 ng/ml FGF8 had no significant effect. (F) RT-qPCR, E12.5 CPCs. SU5402 increases (mRNA) Msx1 levels, whereas the BMPR inhibitor LDN-193189 reduces (mRNA) Msx1 levels and increases (mRNA) Spry1 levels. (G) RT-qPCR, E12.5 CPCs. Like FGF, EGF downregulates Msx1, and the EGFR inhibitor PD153035 increases (mRNA) Msx1 levels. Error bars represent standard errors.(TIF)Click here for additional data file.Figure S2Possible modes of BMP-FGF cross inhibition and behaviors of different CIPF motifs. (A) Three possible ways in which FGFs can inhibit BMP target responses: 1) inhibition directly at the measured target (red), 2) upstream of the target at the level of intermediate messengers (blue), or 3) at the level of second messengers (green). (B) One potential BMP-FGF interaction, which generates a cross-inhibitory positive feedback (CIPF) loop. (C) Effect of FGF addition on BMP target responses for some of the CIPF motifs shown in 50 values, due to suppressed target levels at low BMP concentrations and maintained levels with high BMP. (D) An example of stoichiometric inhibition of BT. The figure shows the effect of increasing BMP concentration on the concentrations of BT bound to FT and unbound BT.(TIF)Click here for additional data file.Figure S3Classification of BMP-FGF cross inhibition models. (A) After simplifying the BMP and FGF signaling pathways to BMP→BI→BT and FGF→FI→FT, there are 81 potential models of cross inhibition between the two pathways: 9 STI, 18 SUI, 18 CFF, and 36 CIPF. The boxed networks denote the models that were chosen as representative models for the B) Topologies of possible CFF (top row) and CIPF sub-motifs (bottom row).(TIF)Click here for additional data file.Figure S4Distinguishing amongst BMP-FGF cross inhibition models. (A) The same four representative models shown in B) Representative BMP dose-response simulations with a nonlinear BMP core pathway (nH = 10 for B→BI or BI→BT) and with or without FGF . See C) BT responses with all links linear (context 1), but with increasing inhibitory strengths . Other parameters match those in D) Schematics of different types of memory; red and blue lines indicate responses that start either on or off, respectively. From left to right – 1) no memory, in which responses do not depend on starting condition; 2) hysteresis, in which responses depend on starting condition, but can return to 0; and 3) irreversibility, in which the response, once ‘on’, never returns to 0. (E) Comparison between CIPF (top row) and auto-regulatory positive feedback (bottom row), with increasing feedback strength from left to right. Increasing CIPF feedback strength increases its bistability window, but never produces irreversibility, as BT can always returns to 0. Auto-regulatory positive feedback can generate irreversibility, depending on feedback strength.(TIF)Click here for additional data file.Figure S5CIPF inhibitory links need to be balanced to produce ultrasensitivity and hysteresis. (A) The CIPF loop with its parameters for strength (p) and linearity (n). (B) Hysteresis, monotone systems analysis. Hysteresis occurs (green dots) when CIPF inhibitory link strengths (left) or nonlinearities (right) are roughly matched. (C) Hysteresis, simulation analysis (varied across a wider fold range to those used for D) Ultrasensitivity, simulation analysis (same parameters as those used to examine hysteresis). Like hysteresis, ultrasensitivity increases mostly occur when CIPF inhibition strengths or nonlinearities are roughly balanced. (E) Comparison of ultrasensitivity and hysteresis increases (bistability window size) for the parameter sets used in (TIF)Click here for additional data file.Figure S650 values and expression domains.Ability of CIPF to generate different EC (A) Immunohistochemistry of adjacent coronal sections of E10.5 Msx1-nlacZ dorsal telencephalon. The Msx1 expression domain detected with anti-lacZ antibody (left) is smaller than the Msx1+Msx2 expression domain detected with an anti-MSX1/2 antibody (right) by ∼200 µm (∼100 µm per side). Scale bar, 0.2 mm. (B) Parameter explorations of BT maximum level and EC50 values with two CIPF networks that are different only in terms of links or degradation rates that influence BT, BI, or FI; a value of 1 indicates that the EC50 or maximum level is same for both networks. Changes to parameters that affect BT do not change EC50 values, while changes to parameters that affect BI and FI in the CIPF loop can produce multiple EC50 values.  Continuation of T EC50 values. Simulated BT response curves represent unchanged (blue) ‘vs’ modified (purple) CIPF networks. (C) Networks in which the balance between BI and FI is changed shift the EC50. These shifts occur upon changing the gain or strength of FI-to-BI inhibition, BI-to-FI inhibition, or the degradation rate of BI. (D) Network changes downstream of the CIPF loop, such as changes in BT degradation rates, do not shift EC50 values.(TIF)Click here for additional data file.Figure S7BMP4 activates and FGF2 inhibits Msx1/2 expression in CPCs at the single cell level. MSX1/2 immunocytochemistry of E12.5 CPCs. (A) With increased BMP4 concentration, more cells express Msx1/2. FGF2 (10 ng/ml) markedly reduces Msx1/2 expression by BMP4 at 1.5 or 16 ng/ml. Scale bar: 0.1 mm. (B) The FGFR inhibitor PD173074 (100 nM) increases Msx1/2 expression in CPCs treated with BMP4 (16 ng/ml) and FGF2 (10 ng/ml). (C) Paradigm for the immunocytochemistry single cell experiment. CPCs were plated and treated with media containing FGF, PD+FGF, or no FGF for 24 hours, at which point BMP4 at 1.5 or 16 ng/ml was added. They were then cultured for 48 hours, fixed, labeled for Msx1/2, then Hoechst counterstained.(TIF)Click here for additional data file.Text S1Supplementary information text. Provides (mostly) mathematical and computational analyses that support the main text. Contents include:Tables S1, S2, S3, S5 listing parameter values of simulations in Table S4: a summary of parameter exploration results shown in Table S6: examples of BMP-FGF inhibitory interactions in development.Table S7: examples of toggle switches in development.Supplementary Numerical Methods and Mathematical representation of biological interactionsMathematical models of BMP-FGF mutual inhibitionGenerating and classifying models of BMP-FGF mutual inhibitionParameter explorations to distinguish BMP-FGF mutual inhibition modelsParameters that enable hysteresis and ultrasensitivityContrasting different mechanisms that generate bistability50 valuesParameter explorations of CIPF subnetworks that can produce multiple EC(DOC)Click here for additional data file.