Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit. Greatwall kinase regulates a switch between kinase and phosphatase activity during mitotic entry and constitutes an essential element of mitotic control. This control system is further complicated by the fact that Greatwall itself is regulated via phosphorylation and acts by phosphorylating its substrates ENSA and ARPP19. A missing link in this central mitotic switch is the phosphatase that counteracts Greatwall and its target Ensa. We have used mathematical modeling and experimental validation to identify these phosphatases. We demonstrate genetic evidence that Greatwall phosphorylation is counteracted by PP2A/B55, while Fcp1 regulates ENSA dephosphorylation. Based on these findings we present a new model for the regulation of mammalian cell division. Phosphorylation of more than thousand proteins by Cdk1 and other mitotic kinases drives entry into mitosis To gain a more precise understanding of this switch-like transition, the phosphatases that target Gwl itself and Ensa/ARPP19 have to be identified. Inactivation of these phosphatases is likely to play a major role in the initiation of the Cdk1 activation loop. Moreover, the reactivation of these phosphatases is likely to be a crucial element during mitotic exit. The identity of the major Cdk1 counteracting phosphatase in mammalian cells is also still under debate. Current models propose that PP2A/B55δ is not only required for the Cdk1 activation loop, but also to directly dephosphorylate mitotic substrates during mitotic exit To gain insight into the dynamics of the Gwl/Cdk1 feedback loop during mitotic entry and exit we performed mathematical modelling  of the Cdk1 regulatory network shown on To test these predictions it is necessary to monitor Gwl phosphorylation during mitotic entry and exit using a phospho-specific antibody. In order to study potential Gwl activation by phosphorylation, we scanned for Cdk consensus sites following the universal DFG motif in Gwl that marks the beginning of the T-loop/activation segment. We also determined the precise localization and timing of Gwl Thr194 phosphorylation by immunofluorescence –D. The pin vivo Cdk1 is required for this phosphorylation. Release from Cdk1 inhibition by 1NMPP1 resulted in rapid simultaneous Cdk1 Tyr15 de-phosphorylation and Gwl Thr194 phosphorylation within 5 minutes  and Cdc25 (Cdk1→Cdc25→Cdk1). Cdk1 subunit is in excess compared to its activating partner Cyclin B. Therefore, it is assumed that all Cyclin B is in the complex such that total CyclinB (CycBT) is sum of active Cdk1/CycB and inactive Cdk1/CycB dimers.The network also includes the regulation of Cdk1/CycB counteracting phosphatase, PP2A/B55. We consider that the dephosphorylation of Wee1 and Cdc25 is controlled by PP2A/B55. Cdk1 indirectly inhibits PP2A/B55 via Gwl-Ensa/ARPP19 pathway. Thus, Cdk1 promote Wee1 and Cdc25 phosphorylations by directly phosphorylating them and by indirectly inhibiting their phosphatase forming coherent feed-forward loops. Gwl-Ensa/ARPP19 pathway involves Cdk1/CycB dependent phosphorylation of Gwl (Gwl) which in turn phosphorylate the phosphatase inhibitor Ensa/ARPP19. The phosphorylated Ensa/ARPP19 binds directly to PP2A/B55 to form an inhibitory complex. The phosphatases that dephosphorylate Gwl and Ensa/ARPP19 are unknown. Therefore, we consider three different scenarios. Gwl is dephosphorylated by (A) OA insensitive phosphatase (B) OA sensitive phosphatase (PP2A) and (c) PP2A/B55. We also assume that ENSAPt (sum of phosphorylated forms of Ensa/ARPP19) is dephosphoryated by OA insensitive phosphatase. We predict using the model the effect of different phosphatases on both the steady state of the system and dynamics of chemical inhibitor induced mitotic entry and exit.http://www.math.pitt.edu/~bard/xpp/xpponw95.html).The temporal dynamics of each component in the network is described by non-linear ordinary differential equation (ODE). All the individual biochemical reactions are approximated by mass action kinetics. The set of ODEs are integrated numerically using stiff solver in XPPAUT, a freely available program from G. Bard Ermentrout, University of Pittsburgh, PA, USA; One parameter bifurcation diagrams Suppl.  were comThe parameter values in XPPAUT code correspond to the situation of Gwl dephosphorylation by PP2A/B55 and the other two models can be simulated with following changes: Gwl dephosphorylation by OA insensitive phosphatase: kigwl′ = 2; kigwl = 0.Gwl dephosphorylation by OA sensitive phosphatase (PP2A): kigwl″ = 2; kigwl = 0.The chemical inhibition of Cdk1 and PP2A are modelled as a reversible inhibitor binding reaction. The mitotic entry simulation in the presence of inhibitor is done with parameter value RO = 25 (for Cdk1 inhibition), OA = 100 (for PP2A inhibition).The mitotic exit simulation in the presence of inhibitor is done with parameter value RO = 100 (for Cdk1 inhibition).# Initial conditionsinit MPF = 0, Cdc25 = 0, Wee1 = 1, Gwl = 0, ENSAPt = 0, PP2 = 0.5# Use the following initial conditions for mitotic exit# MPF = 0.96, Cdc25 = 0.97, Wee1 = 0.03, Gwl = 0.9, ENSAPt = 0.75, PP2 = 0.027#Differential equationsMPF′ = k25*(CycT-MPF) - kwee*MPFCdc25′ = Va25*MPFa*(Cdc25T-Cdc25) - Vi25*PP2a*Cdc25Wee1′ = Vawee*PP2a*(Wee1T-Wee1) - Viwee*MPFa*Wee1Gwl′ = kagwl*MPFa*(GwlT-Gwl) - (kigwl′+ kigwl″*PP2T/(1+OA)+ kigwl*PP2a)*GwlENSAPt′ = kaensa*Gwl*(ENSAT - ENSAPt) - kiensa*ENSAPtPP2′ = -kas*(ENSAPt - (PP2T-PP2))*PP2 + (kdis + kiensa)*(PP2T-PP2)MPFa = MPF/(1+RO)PP2a = PP2/(1+OA)aux preMPF = CycT-MPFaux PP2a = PP2/(1+OA)aux MPFa = MPF/(1+RO)k25 = k25′*(Cdc25T-Cdc25) + k25″*Cdc25kwee = kwee′*(Wee1T -Wee1) + kwee″*Wee1#parameterspar CycT = 1, Va25 = 2, Vi25 = 2, Vawee = 2, Viwee = 2par kagwl = 10, kigwl′ = 0.02, kigwl″ = 0, kigwl = 2, kaensa = 2, kiensa = 0.6par kas = 100, kdis = 1par Cdc25T = 1, Wee1T = 1, GwlT = 1, ENSAT = 1, PP2T = 0.5,par k25′ = 0.01, k25″ = 1, kwee′ = 0.01, kwee″ = 1par RO = 0, OA = 0@ total = 30,dt = 0.5,meth = STIFF@ xlo = 0,xhi = 30,ylo = 0,yhi = 1@ NPLOT = 6, yp1 = preMPF, yp2 = Cdc25, yp3 = ENSAPt, yp4 = PP2a, yp5 = Gwl@ NTST = 15,NMAX = 20000,NPR = 1000,DS = 0.02@ DSMAX = 0.05,DSMIN = 0.001,PARMIN = -1,PARMAX = 1@ AUTOXMIN = 0,AUTOXMAX = 1,AUTOYMIN = 0,AUTOYMAX = 1done5′GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT GGA TCC CAC CGC GGG AAG c3′; 5′GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA CAG ACT AAA TCC AGA TAC GG3′) and cloned into the pCR II-TOPO cloning vector using the TOPO-TA Cloning Kit from Invitrogen. In all cases the manufacturer's recommended protocols were followed and the presence of the expected DNA sequences confirmed by restriction digestion and sequencing. The cDNA was then cloned into an N-terminal Flag tag mammalian expression destination vector . Site directed mutagenesis was carried out using the QuickChange XL Site-Directed Mutagenesis Kit from Stratagene  following the manufacturer's protocol. Human B55α was cloned by RT-PCR as above using the primers 5′GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT A ATG GCA GGA GCT GGA GGA GG3′ and 5′GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA ATT CAC TTT GTC TTG AA3′ cloned into a Gateway Flag-tag expression vector. Fcp1 cDNA was synthesized by Genscript and cloned into the EGFP-C1 vector via Bgl2 and EcoR1 sites. Human Ensa was cloned by RT-PCR using the primers 5′ GGG G ACA AGT TTG TAC AAA AAA GCA GGC TTA ATG TCC CAG AAA CAA GAA GA 3′ and 5′ GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TCA TTC AAC TTG GCC ACC CG3′ and further cloned into a his-tag Gateway bacterial expression vector .Gwl cDNA was cloned by RT-PCR from mRNA isolated from HeLa cells using RNeasy mini kit (Quiagen). Reverse-transcriptase PCRs (RT PCRs) were performed using the SuperScript III One-Step RT-PCR System from Invitrogen. Gwl cDNA was amplified from HeLa cell messenger RNA (mRNA) (using primers Rabbit phospho-(Ser) CDKs substrate antibody was purchased from Cell Signaling. Peptides and polyclonal rabbit Phospho-Thr194 Gwl were generated and purified by Eurogentec, Belgium. Peptides and polyclonal rabbit phospho-Ser67 Ensa/ARPP19 was generated by Generon and we also obtained a phosphor-specific P-Ser67 Ensa antibody from Dr. Satoru Mochida . To make phospho-specific monoclonal antibody (mAB) to phospho threonine and tyrosine on cdk1, a phospho-peptide EKIGEGpTpYGVVYKGC was coupled to KLH and injected into Balb/c mice. Hybridoma cells produced from the spleen cells from a hyperimmune mouse were fused with Sp/0 myeloma cells using standard procedures. Positive clones were identified using immunoadsorbent assays and immunoblotting of Xenopus egg extracts. mAb CP3.2 gave the strongest signal in these assays.; α -tubulin (clone DM1A), myc (clone 9E10) and CENPA (clone 3–19) mouse monoclonal antibodies were purchased from Abcam , PP2A/B55α (clone 2G9) and PP2A/B55 (clone D-10) mouse monoclonal antibodies from Santa Cruz Biotechnology ,PP2AC from Sigma (SAB4200266), histone H3 mouse monoclonal antibody (clone 6.6.2) from Millipore  and mouse monoclonal anti-MASTL from Sigma (HPA027175). HRP-conjugated and polyclonal goat anti-rabbit or mouse antibodies were from Dakocytomation (UK). Secondary antibodies for immunofluorescence were from Invitrogen.2 incubator. Chicken DT40 cells including cdk1as cells were cultured as previously described (24).HeLa cells were cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin in a 37°C, 5% COCdk1as cells were synchronized in G2 with 10 µM 1NMPP1 for 6 h, then were released in mitosis after 1NMPP1 wash out and collected 5 min later for immunofluorescence assays. Alternatively, G2 cells were incubated with 1 µM of OA or/and 10 µM TC for indicated times and harvested for immuno-fluorescence and immuno-blotting analysis.HeLa cells were synchronized with 5 µM of S-trityl-L-cysteine  for ∼18 h, then were harvested by mitotic shake-off. The cells were then incubated in STLC-containing media supplemented with 1 µM of okadaic acid  or/and 10 µM of tautomycetin , or 100 nM of calyculin A  for 1 h. Next, Cdk1 activity was inhibited with 10 µM of RO3306  for 0, 15 or 30 min and the cells were collected for immunoblotting analysis.5′-GCAGAUGAUUUGCGGAUUA-3′ for B55α and 5′-CAUCCAUAUCCGAUGUAAA-3′ for B55δ, Qiagen, Manchester, UK) were reverse transfected using Lipofectamine RNAiMAX reagent (Invitrogen) two consecutive days according to the provider's instructions. The cells were treated with STLC for mitotic synchronization 48 h after the 2nd transfection and mitotic exit experiment was carried out the following day.To knock-down PP2A/B55α and δ subunits, 40 nM of each siRNA B55 ) and 10 µl 5× sample buffer . The samples were sonicated then boiled at 95°C for 5 min. Samples were then analysed by western blotting and the signal was using Immobilon Western Chemiluminescent HRP substrate (Millipore). Image J was used to quantify the intensity of phospho-(Ser) CDKs substrate signal. α-tubulin was used to normalize the samples.2. 24 h later, 5 µM of STLC, 75 ng/mL Nocodazole or 10 µM RO3306 was added to the media and incubated for 16–18 h. Indicated cells were then treated with 1 µM of OA or/and 10 µM of TC or 100 nM of calyculin A for 1 h. Mitotic exit was induced by adding 10 µM of RO3306 for 30 min. Cells were lysed on ice for 20 min in IP buffer ) supplemented with the appropriate phosphatase inhibitors . Protein samples were clarified by centrifugation at 13 000 rpm for 30 min at 4°C. Flag-Gwl protein was captured on anti-flag M2 magnetic beads (Sigma) for at least 2 h at 4°C. After 2 washes with IP buffer and 2 with kinase buffer , the beads were incubated with 10 µg of myelin basic protein  diluted in kinase buffer supplemented with 25 µM of ATP and 0.003 MBq of γ-32P-ATP for 20 min at 37°C. The reaction was stopped by adding the sample buffer and was analysed by western blot and autoradiography. Recombinant Cdk2/cycA was a gift from Dr. Julian Gannon .HeLa cells were transfected with 10 µg of Flag-Gwl plasmid  following the manufacture's guidelines. Hek293T cells were transfected using CaClHeLa cells were grown on coverslip, then were fixed with 3.7% of formaldehyde  in PBS for 10 min  or mitotImages were acquired on DeltaVision microscope equipped with a UPLS Apo, N.A. 1.40, 100× oil immersion objective (Olympus), standard filter sets  and a CoolSNAP_HQ2 camera (Photometrics). Images were exported to Omero software (Version 4.4.4). Deconvolution were performed using SVI Huygens Professional Deconvolution Software (Version 3.5) and images were converted into Adobe Photoshop files.2, 15 mM KCl, 1 mM EGTA, 5 mM NaF, 20 mM β-glycerophosphate, 1 mM DTT and 1.85 MBq γ-32P-ATP (PerkinElmer)). To eliminate unincorporated ATP and the kinase, the phosphor-Ensa protein was captured on Ni-NTA beads. After elution, the phospho substrate was concentrated by spinning on ultrafiltration columns  in buffer containing 20 mM TrisHCl pH 7.5, 150 mM NaCl and 0.01% Tween 20. Hek293 cells were transfected with GFP-FCP1 using a calcium phosphate transfection protocol. 24 h later, cells were treated with 100 ng/mL of nocodazole for 16–18 h and mitotic cells were incubated in DMSO or 10 µM of RO3306 for 30 min at 37°C. GFP-FCP1 was then immunoprecipitated from cell extracts using magnetic GFP-Trap_M beads  according to the provider's instructions. After washes, beads were resuspended in phosphatase buffer . Phospho-Ensa was added to the mixture and samples were incubated for 90 min at 30°C. The reaction was stopped by TCA protein precipitation then the samples were treated as described in Recombinant His-tagged Ensa was purified using Ni-NTA agarose (Qiagen) from BL21 E.coli following the manufacturer's protocol. 1 mg of purified protein was then phosphorylated for 2 h at 37°C by recombinant engineered Gwl kinase  in kinase buffer . The kinase reaction and the purification of the phosphorylated substrate were similar to the preparation of the phosphorylated recombinant ENSA. Flag-B55α was transfected in HEK293 cells. The phosphatase assay with B55α was carried out in a similar way as FCP1 phosphatase assay except the Flag-B55α was captured on anti-flag M2 magnetic beads (Sigma) and phosphorylated his-Gwl was used as a substrate.Recombinant His-tagged Gwl was produced in insect cells and purified on Ni-Agarose beads. and concentrated by spinning on ultrafiltration columns  in 40% glycerol/PBS buffer. His-tagged Gwl was phosphorylated Figure S1Steady state analysis of the mitotic switch. The steady state fraction of Tyr15 Cdk1 phosphorylation and phosphorylated Gwl are plotted as a function of CycB levels, assuming that Gwl is dephosphorylated by an OA-insensitive (A), an OA-sensitive phosphatase (B) or by PP2A/B55 (C). The ‘balance curves’ were calculated in the absence of any inhibitor (green curves), in the presence of Cdk1 inhibitor (red curves) and Cdk1 plus PP2A inhibitors (blue curves). Solid and dashed lines represent stable and unstable steady states, respectively. Down-regulation of the phosphatase on Wee1 and Cdc25 by Cdk1 activity is required to generate bistability in our model. This is achieved through the Cdk1-dependent activation of the Gwl-ENSA pathway that inhibits PP2A:B55 phosphatase. This explains the loss and the persistence of bistability after Cdk1 and PP2A combined inhibition (blue curves) with OA-insensitive  and OA-s(TIF)Click here for additional data file.Figure S2A) WT and Thr194Ser Flag-Gwl were transiently expressed in 293T cells. 48 hours after transfection the cells were synchronized in mitosis by 18 hour incubation with nocodazole. Mutant and WT kinase were immuno-precipitated and assayed for kinase activity using MBP as a substrate. The kinase assays were analyzed by SDS PAGE and autoradiography. (B) FACS profile of thymidine release samples taken at indicated time points after thymidine washout. Cells passed through mitosis between 8 hours and 10 hours, correlating with Gwl Thr194 phosphorylation. (C) siRNA depletion of Gwl to determine specificity of the immuno-fluorescent signal of the phosphor Thr194 antibody. Cells were siRNA transfected with control and Gwl siRNA. 48 hours later, cells were treated with STLC to achieve mitotic arrest and stained with anti Gwl pThr194 (green), anti-αtubulin (red) and DAPI (blue). Note the mitotic cells that lost Gwl Thr194 signal in the knock-down sample. (D) Analysis of Gwl Thr194 phosphorylation by immunofluorescence with anti-Gwl pThr194 antibodies. DAPI staining and centrosome separation was used to identify mitotic cells at various stages.((TIF)Click here for additional data file.Figure S3A) Cells were arrested in mitosis by STLC and pretreated with 1 µM OA and 20 µM MG132 before inactivation of Cdk1 by 50 µM Roscovitine (Ros), 5 µM Flavopiridaol (Fp) and 10 µM RO3306 (RO). Mitotic exit was scored by measuring levels of phosphorylated SP by immune-blots. (B) Three independent experiments as shown in (A) were quantified using Image J. Error bars indicate standard deviation in the 3 data sets. (C) Effect of Cdk inhibitors on Gwl activity measured by IP/kinase assays. Flag-Gwl was transfected in 293T cells. 48 hours after transfection the cells were arrested in nocodazole for 18 hours. Flag-Gwl was immuno-precipitated from mitotic cells and incubated with recombinant Ensa/ARPP19 and γ32P ATP. The kinase assays were analyzed by SDS PAGE and autoradiography. (D) Effects of 1 and 5 µM OA on mitotic exit dephosphorylation. HeLa cells were synchronized in mitosis by Eg5 inhibition using 5 µM STLC and pretreated for one hour with 1 µM and 5 µM OA. Samples were taken for extraction and immunoblot analysis at indicated timepoints following treatment with 10 µM RO3306. (E) Effects of OA and TC on FCP1 phosphatase activity. Ensa phosphatase were performed as described in Mitotic exit triggered by Cdk inhibition. ((TIF)Click here for additional data file.


We describe a new breeding behaviour discovered in emperor penguins; utilizing satellite and aerial-survey observations four emperor penguin breeding colonies have been recorded as existing on ice-shelves. Emperors have previously been considered as a sea-ice obligate species, with 44 of the 46 colonies located on sea-ice . Of the colonies found on ice-shelves, two are newly discovered, and these have been recorded on shelves every season that they have been observed, the other two have been recorded both on ice-shelves and sea-ice in different breeding seasons. We conduct two analyses; the first using synthetic aperture radar data to assess why the largest of the four colonies, for which we have most data, locates sometimes on the shelf and sometimes on the sea-ice, and find that in years where the sea-ice forms late, the colony relocates onto the ice-shelf. The second analysis uses a number of environmental variables to test the habitat marginality of all emperor penguin breeding sites. We find that three of the four colonies reported in this study are in the most northerly, warmest conditions where sea-ice is often sub-optimal. The emperor penguin’s reliance on sea-ice as a breeding platform coupled with recent concerns over changed sea-ice patterns consequent on regional warming, has led to their designation as “near threatened” in the IUCN red list. Current climate models predict that future loss of sea-ice around the Antarctic coastline will negatively impact emperor numbers; recent estimates suggest a halving of the population by 2052. The discovery of this new breeding behaviour at marginal sites could mitigate some of the consequences of sea-ice loss; potential benefits and whether these are permanent or temporary need to be considered and understood before further attempts are made to predict the population trajectory of this iconic species. Euphausia superb), and the higher trophic levels which feed on Krill such as glacial squid (Pleuragramma antarcticum); two species which compose the majority of the emperors diet Recent studies suggest that emperor penguin populations will decline in future decades due to climate change As a breeding platform, stable or “fast” sea-ice is required which forms when the emperors arrive at their breeding locations  and remains unbroken until the chick fledge . If the sea-ice breaks up too early in the season it will result in high chick mortality In recent years satellite observations have improved our knowledge of the emperor penguins breeding distribution Whereas sea-ice is frozen sea-water, ice-shelves are floating glacial ice that has flowed from the land into the sea; where the base of such glaciers breaks hydrostatic equilibrium, the ice-foot detaches from the ground bed and the glacial ice floats. When a single glacier feeds into the sea a glacier tongue is formed, but around the Antarctic coastline it is more common for ice from several glaciers or ice-streams to merge to form an ice-shelf. At their terminus ice-shelves can form ice cliffs, in some place over 60 metres high, although a few tens of metres is more common. Ice creeks often indent the cliff face giving a potential route up onto the ice-shelf itself, or where ice shelves are ablating the ice cliff may be less steep. As sea-ice forms, local weather conditions mean it can be highly variable in extent and duration, and therefore highly susceptible to regional climate change It is at present unclear whether this behaviour of breeding on ice shelves is a new phenomenon associated with recent climate change, or one that has always existed but has not yet been documented. Models of how animals adapt to climatic change exist That emperor penguins can move their breeding site depending upon ice conditions to a more stable location, including onto the top of the ice-shelf itself, means new factors should be incorporated into modelled population trajectories for this species. Whether such factors will provide temporary or permanent relief from the impacts of climate change remains uncertain.The fact that emperors exhibit a previously unknown breeding behaviour, intimates that other less-well known species may also have similar unknown adaptive behaviours that may also offer temporary or permanent relief to the challenges of climate change.The first emperor colony found on an ice shelf was the Barrier Bay colony, 67.22°S, 81.93°E discovered in December 2009 The second colony identified on the top of an ice shelf is the Shackleton Ice Shelf colony. This colony was first located in 2008 A. patagonicus), a species which Larsen would have been familiar with from his sealing trips to South Georgia. It is likely that Larsen’s sighting late in the breeding season indicated a colony in the vicinity. Although exhaustive satellite searches of the sea-ice in the area during previous studies The third breeding location on top of an ice shelf is near the Jason Peninsula, at the northern limit of the Larsen C Ice Shelf. In 1893, the explorer and sealer Carl Anton Larsen was the first to visit this area However, in 2012, a further satellite survey for emperor colonies was conducted along the edge of the Larsen C Ice Shelf. A medium sized colony was discovered on top of the shelf . An aerial survey of the colony was undertaken in early December 2012  revealinFinally, a colony was sighted on top of an ice shelf at the Ruppert Coast colony. This colony was only discovered in 2010 when it was located on the sea-ice under the ice cliffs of the Nickerson Ice Shelf at 75.38°S, 143.35°W. Satellite imagery shows that in 2008, 2011 and 2010 (no images from 2009 are available) the colony was located on the sea-ice at 75.38°S, 143.28°W, but in 2012 (17/10/2012) it had moved onto the edge of the ice shelf above the ice cliff . At presTo assess why the colony location had moved from sea-ice to ice-shelf ENVISAT synthetic aperture radar imagery of sea-ice concentration was acquired of the Shackleton Ice shelf colony (for which the most data existed) several times over the course of the breeding season. The imagery from March, when adults start to return to their breeding location http://www.polarview.aq/), mean temperature was assessed using the RACMO region climate model To test whether the presence of colonies that have been found on ice-shelves was linked to environmental conditions, three environmental variables were assessed at the four sites and compared with values for other emperor penguin colony locations . Autumn Of the four colonies described here, the three that have been found breeding on ice shelves in multiple years could be described as located in marginal conditions. The Shackleton Ice Shelf and Barrier Bay sites have the lowest mean autumn sea-ice concentrations of any colonies and both these and the Larsen ice-shelf colony are in the most northerly part of the emperors range and have higher-than-average mean-temperature regimes. Three other colonies have mean annual temperatures higher than the Larsen colony; Smyley Island, Bowman Island and Snow Hill Island. On Smyley Island in 2009 the colony was observed by QuickBird satellite imagery breeding on top of an iceberg , a possible response to poor sea-ice earlier in the season. A similar behaviour has been observed at the Mertz Glacier colony which is also located in an area that has a high mean temperature (André Ancel pers coms.). This behaviour at these other colonies suggests that breeding on ice-shelves is only one of several possible adaptations that could be employed by emperor penguins when sea-ice conditions are poor; others include moving onto land (for example Dion Islands). Of the other warmer sites the Snow Hill Island colony has not only the warmest mean temperature but is also is the highest latitude, although it has a reasonable high mean sea-ice concentration. Bowman Island has the third highest mean temperature and the fourth lowest sea-ice concentration. These two must be considered some of the most marginal and potentially vulnerable of emperor breeding locations as neither colony has the option of moving onto floating ice-shelves as there are none in the nearby locality and neither has it shown evidence of breeding on icebergs.The Ruppert Coast colony that has been found located on the edge of an ice shelf in one year is neither in a warm or poor sea-ice location; as yet we have no explanation of why this colony moved to a more elevated location.Only a relatively small number of emperor colonies have been studied across Antarctica The phenomenon may be recent phenotypic plasticity as regional climate change affects parts of the Antarctic coastline.Most previous study-sites are not located at ice-shelf breeding colonies where this behaviour is exhibited.There have been no large scale systematic searches for emperors on ice shelves .Visser The ability of emperor penguins to change their breeding platform when fast ice conditions deteriorate may be an important adaptation that could help the species survive in a warming environment. Although regional warming has led to loss of ice-shelves around the Antarctic Peninsula For emperor penguins, the loss of the sea-ice as a breeding platform is not the only consequence of a warming environment. Factors, such as changes to food webs Currently, sea-ice conditions and their predicted decline are one of the key inputs into models that suggest large decreases in emperor penguin numbers. Therefore, the suitability of ice-shelves versus sea-ice as a breeding platform for emperors urgently needs quantifying. Future research efforts need to assess which colonies could potentially move to ice-shelves and whether this newly described behaviour is a temporary or partial solution for coping with future climate change. Emperors are often portrayed as a barometer for the ecosystem, that is, a “canary in the coalmine” for species more difficult to study. This previously unknown and surprising behaviour recorded in such an iconic animal suggests that other species may also be capable of unpredicted or unknown behavioural adaptations that may also increase their survival in a future warming world.


Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system.Extensive variation in early gap gene expression in the Drosophila embryo, we investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, we analyzed attractors and their basins of attraction in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. We showed how the border formation is dictated by the biological initial conditions  being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets  define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. We demonstrated how the positional variability for hunchback is related to the corresponding variability of the basin boundaries. The observed reduction in variability of the hunchback gene expression can be accounted for by specific geometrical properties of the basin boundaries.In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryos. These mechanisms were specified in the case of hunchback in well defined terms of the dynamical system theory.We clarified the mechanisms of gap gene expression canalization in early  Development is surprisingly robust to environmental stress, intrinsic fluctuations, and genetic variability in populations. These facts, together with the observation that cell type is a discrete rather than continuous property, led C. H. Waddington to propose that developmental processes have innate error-correction properties, which he called "canalization" . WaddingDrosophila melanogaster [even-skipped stripes in three lines and two species of Drosophila [We recently demonstrated the existence of canalization at the molecular level in the segment determination system of the fruit fly nogaster . We shownogaster , and furnogaster , a precinogaster . That wonogaster -8. In a osophila  dependedosophila  because osophila .In comparing our results to those of Lott and coworkers, we note that we considered the canalizing behavior of a specific zygotic component of the segmentation system, while the study of Lott et al. looked at the canalizing behavior of the full set of organismal genes, many subsets of which are presumably engaging in their own form of canalizing behavior. Closing the gap between the genetic control of canalization at the level of populations versus the well defined small networks of genes considered in developmental genetics will require finding the specific genes responsible for population effects as well as taking the complementary step of incorporating additional mechanisms and genes into the well characterized systems arising in a developmental genetics context.in silico. Our model accounts for the expression of four mutually interacting gap genes, hunchback (hb), Krüppel (Kr), giant (gt), and knirps (kni), and takes into account the expression of the genes bicoid (bcd), caudal (cad), and tailless (tll) as external inputs. Parameter values in the model were calculated by fitting solutions to mean time dependent expression levels from zygotic and maternal/zygotic genes and bcd expression from a single embryo. The resulting model, when run with Bcd gradients from many individual embryos, correctly predicted the variance in position of six gap gene borders. Analysis of model behavior at the numerical level showed that the observed variance was a consequence of gap gene cross regulation [In ,4, we usgulation . To furtThis analysis showed that the observed reduction in variation of gap gene expression patterns is a consequence of the action of robust attracting states . The forThere were two limitations to this analysis. First, the dynamical interactions underlying the observed reduction in variance were elucidated by performing the dynamical analysis on one particular circuit controlled by the median Bcd gradient used for the fit. As a consequence, the variance reduction analysis described in  was shifhb border formation as an example. We analyzed how this border forms in terms of the phase portrait of the gap gene dynamical system for various Bcd profiles. We find two specific mechanisms responsible for canalization of the variance, both connected to the phase portrait geometry.The key idea of this extended analysis is to express gene expression variability in terms of such basic objects of the dynamical systems theory as attractors and attraction basins. The canalization will be explained by specific geometrical properties of these objects. We demonstrate the applicability of this approach using in vivo work that our profiles exaggerate variance in Bcd amplitude. However, because there is no independent reason to believe that background should be adjusted to minimize variance, the in situ data processed in this manner should be viewed as a lower limit on embryo to embryo variation of the Bcd profile.Two sets of spatial profiles of the Bcd concentration were obtained by the numerical processing of raw Bcd data from individual embryos in two different ways. One set was identical to that previously described : the BcdvBcd(x) = A exp(-lx) with x varying along the A-P axis of the embryo. We obtained in this way a set of 88  Bcd parameters {A, l}  in nucleus i (1 ≤ i ≤ M(n)) along the A-P axis of the embryo. The model incorporates nuclear divisions, number of nuclei M(n) and diffusion coefficients aD(n) depend on the cleavage cycle number n. The function a Rsynthesis of the ath protein. The argument u of g contains inputs from various transcriptional regulators. The first input is a linear combination of all abT, which thus quantify the regulatory interactions between the genes. The Bcd concentration profile χ(t) equals one during interphase and zero during mitosis, accounting for the fact that synthesis shuts down during this period. Coefficient i = 1 (i = M) and one otherwise, preventing protein diffusion outside of the spatial domain. Coefficient a λis the rate of protein degradation, and constant a hadjusts the threshold of the regulation function.where The initial conditions in the model consist of a spatial gradient of maternally expressed Hunchback (Hb) protein concentration and zero concentrations of the other three proteins. These conditions correspond to the state of the gap gene system at the start of cleavage cycle 13. The solutions in the model are biologically meaningful until the end of cleavage cycle 14A, at which time the midblastula transition occurs and many properties of the embryo change. The nuclei range along the A-P axis within the spatial domain from 35% to 92% of the embryo length (EL) where the gap genes express [All gene expression levels ier work . The parier work  were thoier work ). For thier work ,20.M model equations to M independent systems of four equations, each system corresponding to one nucleus. We investigated only a restricted region of the A-P axis surrounding the posterior border of the anterior hb expression domain. The region ranges from 37% to 57% EL, about 11 nuclei in width in cleavage cycle 13 and 22 in cleavage cycle 14A. Tll does not act in this region and can be omitted [We considered a simplified version of the model without diffusion, called the "shorted model" as in previous work , keepingx indicates the parametric dependence of solutions. It stems from the dependence of Bcd and Cad protein concentrations on spatial position as determined from experimental data. Such dependence can come directly from experimental data in nuclei, for which x = i, or as a continuous real-valued interpolation x.where index vCad from the beginning of cleavage cycle 13 to the end of time class T6. Thereafter we take vCad = vCad and χ(t) = 1 for times t >T6. Thus, the shorted equations (2) are nonautonomous until T6 and autonomous later.The earliest observable indications of the mid-blastula transition, which include decay of the Bcd gradient, are evident after time class 6 (T6) of cycle 14A, about ten minutes prior to the onset of gastrulation ,4. For tx from 37%-57%EL region in two mutually complementary directions. We performed a bifurcational analysis in the autonomous version of the system (2), in which χ(t) ≡ 1 and vCad was replaced by value vCad. The bifurcation structure in the system was studied on the Bcd-Cad plane, which is the plane with coordinates vBcd and vCad (with vCad corresponding to the Cad concentration at t = T6), by means of the AUTO package [vHb ≤ 100, vKr = vGt = vKni = 0} . Each point attractor is the asymptotic limit at late times of dynamics starting from the initial conditions which are grouped in a certain part of Ω, and we call this part the basin of attraction for the attractor. To get a more spatially refined picture, we also performed a calculation of attraction basins at thirty spatial positions in the range 37%-57%EL for the Bcd ensemble normalized by the basic method.We analyzed the dynamical system (2) for spatial position  package . In thisx, the attraction basin of each attractor can be represented as an interval (c1(x), c2(x)) on the Hb axis, as all other protein concentrations are zero in Ω. The values vHb = c1 and vHb = c2 are lower and upper boundaries for the basin, respectively. We used linear spatial interpolation for these values in order to study the attraction basin boundaries as continuous functions of x for each Bcd profile. Some attraction basins consist of more than one disjoint interval on the Hb axis. In this case, the basin boundaries comprise the boundaries of each connected part of the basin.For each Bcd profile and fixed spatial position S having a single eigenvalue with positive real part by solving the simplified model equations from two initial conditions S ± w, where w is a scaled eigenvector  of the Jacobian at the saddle.We calculated approximations for 1D unstable manifolds of saddles hb border positions in solutions exhibited at the onset of gastrulation, we used spatial interpolation of first and third order for solutions in the shorted and full models, respectively. The orders are different for the two models since the solution in the shorted model is less smooth in space than in the full one because of the absent diffusion term, and the use of high order interpolation schemes could lead to artifacts. As a consequence, we computed the border positions in different ways for the two models. The hb border position in the spatially interpolated solution of the shorted model was defined as the point in the spatial domain at which Hb concentration reached its half-maximal value, and in the full model as the local inflection point.In order to calculate the We extend the dynamical analysis reported previously for the median Bcd profile  to the ex denoting A-P positions in the range 37%-57% EL or a point on the Bcd-Cad plane , either an explicit parameter e Figure . We furtKr and gt in the anterior vicinity  from 35% to 71% EL occur in the same order and locations as those generated by the full model equations (1). The only exception is that the borders become very sharp and domains tend to be mutually exclusive . Over t from 35%vHb ≤ 100, vKr = vGt = vKni = 0}.For each Bcd gradient, we performed an analysis of how border formation was driven by dynamical attractors. Because different Bcd profiles lie on different portions of the Bcd-Cad plane, we first characterized which combinations of attractors are present in different parts of this plane by performing a bifurcational analysis Figure  of the sA1-A4) in the portion of the Bcd-Cad plane shown in Figure A3 = 0100 means that Kr is highly expressed at this attractor with the other genes staying repressed. A1 is the only attractor that continuously changes its code with varying Bcd and Cad concentrations. There are six domains on the Bcd-Cad plane in which various combinations of A1-A4 exist  are very close to point attractors in the anterior regime and to attracting manifolds in the posterior one ; the transition in Figure A2 to the basin of A1 (A2 → A1 transition). The basin-to-basin transitions can also be detected in Figure hb-expressing and hb-nonexpressing border nuclei eventually end up at the same point attractor A2. The hb border forms in this case according to the mechanism from Figure hb-nonexpressing border nucleus is attracted to a hb-OFF state on an unstable manifold which connects a saddle and attractor A2 (data not shown). This Bcd profile was excluded from the further analysis. For all cases with the A1 → A3 transition, attractor A1 is in the state hb-expressing border nucleus. The A1 → A3 and A2 → A3 transitions are similar in the sense that they both correspond to the switch from an attractor with hb-ON state to an attractor with hb-OFF state. We found 69 Bcd profiles in the ensemble associated with either of these two transitions. We call these profiles Family I in what follows. These transitions can hypothetically correspond to any of the four mechanisms possible for the hb border formation, because the attracting invariant manifolds may also participate in the border formation in this case . One can speculate that these two positions are the manifestation of two levels of positional information readout in the gap gene circuit. In this subsection, we compare the positional variability for hb predicted by the model for these two levels. We calculated the spatial profiles of the basin boundaries for all Bcd gradients. The ranges of the border positions corresponding to the two Bcd families are shifted with respect to each other, and the same shift is observed at the level of the basin boundaries  shifts of the hb border for some range of Bcd concentration in the presence of gap gene cross regulation . We studied the model on the newly normalized Bcd profiles in order to crosscheck our results. The corresponding parameters σ = 1.2%EL. The corresponding positional variance for hb in the full model equals to σ = 0.5%EL.Due to the specific statement of the alternative normalization method, the Bcd positional variance in the ensemble became much lower: the standard deviation A5 = 0101 and A6 = 1000, in addition to attractors A1-A4 which are analogs of the corresponding attractors to the above study. Attractor A6 exists in a very narrow domain on the Bcd-Cad plane, where it coexists with attractor A1 at the state A4, since the newly normalized Bcd concentrations never entered the existence domain for this attractor  . It turns out that the hb border formation mechanism involving the attracting manifolds is mostly associated with these cases. This may lead to the conclusion about restricted applicability of this mechanism in the case of hb expression. However, this mechanism exists and plays an important role for the gap domain borders in a posterior part of the embryo, where the domains form and vary in time under the control of an unstable manifold [huckebein [The manifold . To analuckebein .hb border formation [hb border position is well encoded by the intersection between the initial Hb profile and corresponding attraction basin boundaries, the stability of hb border predicted by the model can be explained by inspecting the geometrical properties of these attraction basins.As previously reported, the model exhibits a significant filtration  of the Bcd positional variability at the level of ormation -4. Our rormation ,10 that ormation ,24. We tpurely geometrical fact evidently prescribes a smaller variation of the intersection point when the basin boundary is changing due to the variance of Bcd concentration, as opposed to the case if the curves would jointly rise or jointly fall along the A-P axis  to study the canalization mechanisms based on the assessment that the model provides one of the best spatio-temporal precision for the description of gap gene expression . This mohb border position in the model. The nonlinear part of the curve can be explained by the regulatory actions on hb from the other gap genes. In particular, a regulatory analysis in the full model revealed that the regulatory interactions between hb, gt, and Kr underlie the folding part of the response curve .Click here for fileThe bifurcation diagram with more details.Click here for fileA1-A4 following from calculations at discrete spatial positionsThe existence domains on the Bcd-Cad plane for attractors .Click here for filehb border formation and over solution classes I-IIIThe distribution of Bcd profiles over the four mechanisms of .Click here for fileThe response curve for the normalized individual Bcd profiles instead of their exponential approximations.Click here for fileNew parameter values in the model obtained by optimization with a median Bcd profile from the Bcd data normalized by the alternative method.Click here for fileThe solutions of the full model equations and their simplified version for the new parameter values.Click here for fileThe bifurcation diagram for the new parameter values with more details.Click here for fileDetailed description of the bifurcations for the new parameter values .Click here for fileA1-A6 in the model with the new parameter values following from calculations at eleven spatial positionsThe existence domains on the Bcd-Cad plane for attractors .Click here for fileClassification results for the Bcd profiles in the case of the alternative normalization method.Click here for fileThe spatial configuration of attraction basins in the model with the new parameter values.Click here for fileThe response curve for the new parameter values.Click here for fileSchematic illustration of the first canalization mechanism.Click here for fileThe regulatory analysis of the response curve .Click here for file


Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions.Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of 2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.In the present work we demonstrate that after transfer to low COThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen. C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs Carbonic anhydrases (CAs) serve different functions in the metabolism of algae and plants. Their role in carboxylation/decarboxylation reactions made them essential in algal carbon-concentrating mechanisms, ion transport, pH homeostasis and in the production of carbon skeletons by mitochondria  showed impaired water-splitting ability et al.2 evolution by locally providing HCO3− as proton carrier to remove protons from the Mn complex.Cah3, identified in 2-concentrating mechanism (CCM). In fact, all the mutants lacking Cah3 grow photo-autotrophically on high-CO2 levels but exhibit extremely poor growth under low-CO2 conditions cia3, can be complemented by the wild type Cah3 gene However, it has also been suggested that Cah3 is functioning in the CO2, even at the low pH of the lumen, is at least 300 times too slow to support the observed high rates of photosynthesis. He postulated that the lumenal CA could be involved in accelerating the conversion of HCO3− to CO2 in the lumen and thus stimulating the carboxylase activity of Rubisco 2 supply for Rubisco, Hanson et al.2 supply for Rubisco was the primary role of Cah3. Their results demonstrated that, after a short-term exposure to low CO2 conditions in the cia3 mutant, CO2 fixation was already compromised whereas the same short exposure to low CO2 did not directly affect PSII activity According to Raven in vivo phosphorylation depends on Stt7 On the other hand, the essential role of phosphorylation of proteins within the photosynthetic membranes for balancing the light distribution between PSII and PSI in plants and algae is now well established et al.2; their phosphorylation required reduction of electron carriers in the thylakoid membrane, but was not induced by light and both proteins were phosphorylated in the low-CO2-exposed stt7 deficient mutant. The latter was suggested as an early adaptive and signaling response of Chlamydomonas to the limited environmental inorganic carbon.Turkina 2-responsive regulation of Cah3. We show here that this lumenal CA is phosphorylated during acclimation of C. reinhardtii cells to low CO2 conditions. We also provide strong evidence that phosphorylation of Cah3 causes activation of the enzyme and its redistribution to the PSII-devoid thylakoid membranes in the pyrenoid. This is the first report of a CA being post-translationally regulated via phosphorylation and describing phosphorylation events in the thylakoid lumen. We conclude that this mechanism allows algal cells to regulate the photosynthetic reactions by controlling the availability of HCO3−, not only at the early steps taking place in the water oxidizing complex In this study we have characterized the biochemical processes involved in the CO2 concentration is regulating the lumen located Cah3 during growth of C. reinhardtii cells, we followed the changes in both activity of Cah3 and expression of the cah3 gene after transferring cells from 5% (high CO2) to atmospheric CO2 levels . When high-CO2-grown cells of C. reinhardtii were transferred to low CO2 conditions, the lumenal CA activity increased   was affected by the addition of the inhibitor. These results indicate that the activation of the lumenal located CA is, at least partly, under the control of phosphorylation/dephosphorylation mechanisms.Phosphorylation/de-phosphorylation modifications of PSII polypeptides in the thylakoid membranes are now well established ctively) . Neithermic Cah1 , only ex2 conditions by using phosphothreonine (Thr(P)) antibodies. This immunological approach has been introduced in the late 90s as an alternative tool to study in vivo phosphorylation processes which allows overcoming the limitations to detect changes in the level of endogenous phosphorylation of other currently used methods C. reinhardtiiChlamydomonas, Cah3 is peripherally associated with the lumen side of the thylakoid membranes and can be released from the thylakoids using treatments that will not cause the release of the integral thylakoid phosphoproteins In a first approach we analyzed the changes in the phosphorylation pattern of thylakoid membranes upon acclimation to low COChlamydomonas cells either grown under high-CO2 conditions or acclimated to low CO2 for 1 and 2 h. Immunoblot analysis shows that Cah3 was recovered in all of the supernatants .The phosphorylated 30-kDa, protein was immunoprecipitated using affinity-purified antibodies against the Cah3 polypeptide . In addiChlamydomonas that is phosphorylated upon acclimation to low CO2 conditions.To obtain evidence that the cross-reaction of the Cah3 polypeptide with the Thr(P) antibodies was due to a true phosphorylation of this protein rather than to a possible unspecific cross-reactivity, we studied the reversion of the phosphorylation signal following treatment with alkaline phosphatase (AP). 2-grown cells, immunogold particles were localized to the intra-pyrenoid and stroma thylakoids  of the Cah3 is localized to the non-pyrenoid thylakoids in those cells . However, a significant portion of the protein was still associated with stromal thylakoids even under low CO2 conditions.Immunogold labeling experiments show that Cah3 was enriched in the PSII-depleted intrapyrenoid thylakoids in low-CO2-grown Chlamydomonas cells 2 cells was also associated with PSII core complexes, we isolated these complexes from cells growing under low CO2 conditions for 4 h. Immunoblot analysis shows that Cah3 was still enriched in PSII core complexes when compared to thylakoids and PSII membrane fragments, as it occurs with D1 protein of PSII core complexes used as control. These results indicate that the phosphorylated form of the Cah3 polypeptide was not associated with the PSII core complex.It is interesting to note that no cross-reactivity of Thr(P) with any phosphoprotein at 30 kDa was observed in the peripheral fraction of PSII core complexes from both high- and low-COC. reinhardtii cells to low CO2 conditions. Phosphorylation of Cah3 causes partial activation of the enzyme and its redistribution to PSII-depleted thylakoid membranes. Like other thylakoid phosphoproteins, Cah3 seems to be phosphorylated at threonine/serine residues Arabidopsis thaliana  when cells are growing under high CO2 conditions 2 limiting conditions, the Cah3 protein is strongly phosphorylated  Unknown Expressed Protein is encoded in the genome of C. reinhardtii). UEP was found phosphorylated at a serine residue. Multiple phosphorylation of Lci5 may occur at three threonine and four serine residues. Phosphorylation of both proteins occurred strictly at limiting CO2 and it required reduction of electron carriers in the thylakoid membrane, but was not induced by light changes (state transitions). Moreover, both proteins were phosphorylated under low-CO2 in the Chlamydomonas stt7 mutant deficient in the light-activated protein kinase Stt7. The latter was suggested as an early adaptive and signaling response of Chlamydomonas to the limited environmental inorganic carbon and correlates with the pattern of phosphorylation found for Cah3 in this work.As it was mentioned above, acclimation to limiting COThe photosynthetic apparatus is more and more understood as a flexible molecular machine that can acclimate to metabolic and light fluctuations in a matter of seconds and minutes 2  in functional PSII reaction centers, an increase in the activity of PSI versus PSII and an increased cyclic electron transport around PSI It is intriguing why a protein, which is required for the WOC to function optimally under high CO2 evolution rates of PSII core complexes. It is interesting to note that this decrease is of the same order as has been previously reported 2-grown cells resembles those, isolated from the mutant cia3, which lacks an active Cah3, and they required bicarbonate for reaching maximum activity 3− requirement in PSII preparations from low-CO2-grown cells is much smaller than in preparations from the mutant cia3, where up to half of the WOCs were non-functional  which is regarded as the standard wild type in photosynthesis studies, was obtained from the Chlamydomonas Culture Collection at Duke University, Durham, NC, USA. The cell wall-deficient, high-CO2-requiring cia3 double mutant was kindly provided by J. V. Moroney  2/s supplied from cool, white fluorescent lamps. Cells were cultured in minimal medium 2 (high-CO2-grown cells). For experiments in which cells were shifted from high CO2 to low CO2 (0.035% CO2), cells were previously cultured in high CO2 conditions and then shifted to ambient CO2 for various times (low-CO2-grown cells).2-grown Chlamydomonas cells according to Berthold, Babcock and Yocum PSII membrane fragments) were isolated as previously described from higher plants Thylakoid membranes were isolated from high- and low-CODifferential extraction of Cah3 from thylakoid membranes was performed by salt washing. Thylakoid membranes were resuspended to a chlorophyll density of 1 mg Chl/ml in incubation buffer containing 50 mM Tris-HCl buffer pH 8.0, 1 mM EDTA, and supplemented with 1 M NaCl. Samples were incubated in the washing solution for 1 h at 4°C in darkness with gentle stirring. After incubation, samples were centrifuged. The pellet was washed twice with buffer containing 50 mM Tris-HCl buffer pH 8.0, 400 mM sucrose, and 10 mM NaCl, and then resuspended in the same buffer. The supernatants were desalted by serial dilution with the salt-free buffer by centrifugation in Centriprep YM-10 tubes (Millipore). Each desalted sample was then concentrated 15 times by centrifugation in the same tubes overnight. All buffers were supplemented with 10 mM NaF, a protein phosphatase inhibitor.in vivo analysis of the phosphorylation pattern of extrinsic proteins, thylakoid membranes were incubated in medium containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 0.05% (v/v) Triton X-100. After 20 min incubation on ice, the membranes were pelleted and the supernatants were analyzed by SDS-PAGE and western blots.PSII hydrophobic and hydrophylic proteins were extracted from PSII core complexes using a chloroform/methanol  mixture The fraction obtained after treatment of thylakoid membranes with incubation medium containing 0.05% Triton X-100 (100 to 150 µg protein) was incubated for 10 min at 30°C. After this incubation, the sample was divided into two halves. One was treated by adding 30 units of alkaline phosphatase  for 15 min at 30°C; the other was used as a control. The reaction was terminated by adding an equal volume of 2× SDS-PAGE sample buffer. The completion of the dephosphorylation reaction was checked by analyzing samples by western blot.2 cells were diluted to 300 µl by using ice-cold immunoprecipitation buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2% (v/v) Igepal CA-630, and a protease inhibitor cocktail . 10 µl of affinity-purified anti-Cah3 antibodies were subsequently added, and samples were mixed by gentle shaking for 2 h at room temperature. 20 µl 10% (v/v) protein A-Sepharose CL-4-B beads (Sigma) were added, and samples were mixed for 4 h at room temperature. Protein A beads were collected by centrifugation and washed twice with 1 ml washing buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% (v/v) Igepal CA-630, 0.1% (w/v) SDS. The beads were boiled for 5 min in 2× SDS-PAGE sample buffer and samples analyzed by western blot.Samples obtained after washing thylakoid membranes from low-CO2-saturated distilled water 0/tc-1) 10, where t0 was the time for the pH change with buffer controls and tc was the time obtained when CA-containing samples were added.The activity of CA was potentiometrically determined by measuring the time for the pH to decrease from 8.2 to 7.4, at 2°C, in a sample of 2 ml of 75 mM phosphate buffer (pH 8.2), containing 1 mM EDTA and 0.1 mM dithiothreitol (DTT), upon addition of 2 ml of ice-cold CO2 evolution was measured at saturating white light and 25°C using a Clark-type oxygen electrode . Cells were resuspended in 20 mM CO2-free buffer HEPES-KOH buffer (pH 7.2) at a chlorophyll density of 2–5 µg Chl/ml. The cell suspensions were then placed in the illuminated electrode chamber and allowed to consume the dissolved Ci of the medium and the intracellular Ci pool until no net photosynthesis was observed. HCO3− at the indicated concentrations was added and the rate of O2 evolution measured.Photosynthetic O3Fe(CN)6, and 1 mM DCBQ. The light-saturated rates of oxygen evolution in PSII core complexes were measured in a buffer containing 25 mM MES-KOH (pH 6.5), 300 mM sucrose, 10 mM NaCl, and 50 mM CaCl2, in the presence of 1 mM DCBQ and 1 mM K3Fe(CN)6 as described by The light-saturated electron flow through PSII in BBY preparations was measured in a buffer containing 20 mM MES-KOH pH 6.5, 400 mM sucrose, and 35 mM NaCl, in the presence of 1 mM KIsolated thylakoid membranes, PSII membrane fragments, total cell proteins, and PSII core complexes were separated on 12% SDS polyacrylamide gels For immunolabeling experiments, cells were fixed with 3% freshly depolymerized paraformaldehyde and 0.3% glutaraldehyde in PBS for 1 h, transferred to 3% paraformaldehyde and stored at 4°C over night. The pellets were infused with 20% polyvinylpyrrolidone in 2.3 M sucrose in PBS for 3 h, mounted on stubs and subsequently frozen in liquid nitrogen. Ultrathin sections were cut and mounted on formvar coated niquel grids.Preincubation of sections was performed in a solution of 0.01% blocking reagent (Boehringer Mannheim) and 0.75% glycine for 15 min. The sections were incubated for 1 h with the primary antibodies (1∶200 dilution) or with preimmune serum diluted similarly as a control, washed for 30 min in PBS/blocking reagent, and incubated with goat anti-rabbit gold conjugate (Biocell) (diluted 1∶50) for 1 h. Finally, the sections were washed in PBS and distilled water and embedded in 2% methyl-cellulose and 0.3% uranyl acetate.Evaluation of immunogold labeling was made using the computer program UTHSCSA Image Tool version 3.0 . The gold particles associated with the pyrenoid were counted, as were the particles in the same size area in the cytosol and stroma regions of the individual cells.cah3 gene, RT-PCR assays were performed. Total RNA was isolated from 2 ml culture by using the Trizol™ reagent according to the manufacturers protocol . 2,5 ng total RNA from wild-type and mutant cia3 were preheated to 70°C for 20 minutes prior to the RT-PCR reactions. Advantage One-step RT-PCR Kit  was used and 10 µl of each reaction mix were run on a 1% agarose gel. The following primers were used for the different transcripts: tubB2, 5′–CTTGTTCTGCACGTTCAGC–3′ and 5′–AAGCAGATGTCGTACAGG–3′; cah3, 5′-GTTCATTGGCAACATGGAGC-3′ and 5′-TGGCGTATGTCTTGTTGTCG-3′.To determine specifically the relative transcript levels of the Figure S12-grown Association of Cah3 polypeptide with PSII core complexes from high- and low-COC. reinhardtii cells. Immunoblot analysis of thylakoid membranes (Thy), BBY preparations (BBY), and PSII core complexes (Core) from cells of C. reinhardtii with antibodies raised against the over-expressed Cah3 polypeptide (Cah3) and D1 protein of PSII. The lanes were loaded with 10 µg protein.(TIF)Click here for additional data file.


There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium  or in reconstituted medium . The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation. Fertilization is the process by which two haploid gametes, the sperm and the egg, unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosomal exocytosis, sperm penetration through the zona pellucida, and fusion of the gamete plasma membranes in vitro by incubating the sperm in an appropriate culture medium. The in vitro study of capacitation has shown this process to be a combination of sequential and parallel events, which occur both in the sperm head (preparation for the acrosome reaction) and tail (hyperactivation). Recently, capacitation has been divided into the following processes: a) fast and early events that comprise activation of the vigorous and asymmetric movement of the flagellum, which occurs as soon as the sperm leave the epididymis; cholesterol loss from the plasma membrane Capacitation normally occurs in the female genital tract; however, it can also be achieved Post-translational modifications through the phosphorylation of serine/threonine (Ser/Thr) and/or tyrosine (Tyr) residues by protein kinases (PKs) and/or the dephosphorylation of these residues by protein phosphatases (PPs) have a central role in many cellular processes. Mature sperm are transcriptionally inactive, unable to synthesize new proteins. Therefore, the need for these cells to alter their function through protein phosphorylation/dephosphorylation is higher than that of other cell types. Protein phosphorylation, specifically Tyr phosphorylation, is known to regulate sperm motility and capacitation in many mammalian sperm Generally, PPs are classified into two families: serine/threonine phosphatases (PPPs) and phosphotyrosine phosphatases The aim of the present study was to characterize the role of the Ser/Thr PPs PP1, PP2A, and PP2B during human sperm capacitation. We determined which PPs are present in human sperm; characterized their enzymatic activities during capacitation; and, using various inhibitors, evaluated their role in the capacitation process.The research presented in this manuscript was approved by the Ethics Committee on Scientific Research of the University of Antofagasta. The institutional review board approved the use of all human semen samples described in this study. All donors signed a consent form for the use of their sperm cells for research purposes.The following reagents were purchased from Sigma Chemical Co. : Nα-tosyl-Lys chloromethyl ketone HCl (TLCK); bovine serum albumin ; 1,4-diazabicyclo[2.2.2]octane (DABCO); HEPES; dimethylsulfoxide (DMSO); 7-chlortetracycline hydrochloride (CTC); poly-L-lysine hydrobromide; Ponceau red; ethylenediaminetetraacetic acid (EDTA); ethylene glycol tetraacetic acid (EGTA); Hoechst 33258 (H258); Igepal CA-630 (NP-40); phenylmethylsulfonyl fluoride (PMSF); sodium dodecyl sulfate (SDS); leupeptin; calmodulin; and bestatin A. The serine-threonine phosphatase inhibitors okadaic acid, deltamethrin, and calmodulin were obtained from Calbiochem Corporation . Endothall was purchased from Affinity Research Products . The protein phosphatase inhibitor-2 (I-2) was purchased from New England Biolabs . The chemiluminescence detection kit was purchased from Amersham Pharmacia Biotech . Immobilon P transfer membrane was obtained from Millipore Corporation . The DC Protein Assay was obtained from BioRad Laboratories, Inc. . Vectashield was purchased from Vector Laboratories, Inc. .The following primary antibodies were used: anti-phosphothreonine (42H4) n° 9386 ; anti β-tubulin ; anti PP1γ (C-19) ; anti-PP2Bα (482–494); anti PP2A/A  ; and PP2A antibody sampler kit, n° 9780 . The following secondary biotinylated antibodies were used: goat anti-rabbit, goat anti-mouse, and rabbit anti-goat .The basic medium used for all the experiments was a modified Tyrode's medium  BSA. This medium was called Capacitating Medium (CM). The Non-Capacitating Medium (NCM) was similar to CM, except for the lack of BSA and NaHCO3, and the addition of 10 mM NaCl and 10 mM HEPES to maintain osmolarity and pH 6 cells/ml and incubated at 37°C and 5% CO2 in air for different periods between 0 and 18 h, depending on the experimental condition. At the end of each incubation time, sperm aliquots were washed twice in PBS  by centrifuging at 800×g for 5 min. The resulting washed sperm pellet was resuspended in homogenization buffer  at a concentration of 30×106 cells/ml Freshly ejaculated sperm from healthy volunteers were obtained by masturbation after 2–5 days of sexual abstinence. All semen samples were normal according to World Health Organization criteria The sperm capacitation status was assessed using the CTC fluorescence assay method, as described previously SDS-PAGE was performed using 12% gels according to the method of Laemmli 6 cells/ml) were gently smeared onto a microscope slide coated with 0.1 µg/ml of poly-L-lysine and allowed to air dry. Slides were fixed in 4% paraformaldehyde in PBS for 15 min, followed by 3 washes with PBS and permeabilization with 0.1% Triton X-100 for 15 min at room temperature. Then, the slides were incubated for 1 h with a blocking solution containing 2% glycine, 1% BSA, and 50 mM NH4Cl in PBS. The slides were then washed 3 times and incubated overnight with different antiphosphatase antibodies at 4°C. Following incubation, the slides were rinsed with PBS and incubated for a further 1 h with goat anti-rabbit antibody conjugated to Alexa Fluor® 633 or 555. For each primary antibody used, a control slide in which the cells were incubated only with the second antibody was processed and analyzed. Slides were washed with PBS and mounted with Vectashield mounting solution. Slides were assessed by epifluorescence ultraviolet microscopy at 630 and 570 nm wavelengths with 100×oil objective lens magnification to determine the localization of detected phosphatases.Aliquots of sperm cells  For PPs activity assays, we used a non-radioactive method, the “Rediplate 96 EnzChek Serine/threonine Phosphatase assay kit” (R33700) from Molecular Probes, according to the manufacturer's instructions. Briefly, 50 µl of sperm lysate was added to each well that contained the Ser/Thr phosphatase substrate  The kinetics of capacitation and phosphatase activity were analyzed by a one-way analysis of variance with Tukey's post test. Capacitation data in CM, NCM or RCM were analyzed by a two-way analysis of variance with Bonferroni correction. Other data were analyzed by one-way analysis of variance and the Student-Newman-Keuls multiple comparison test for unequal replicates using the Prism program. A difference of P<0.05 was considered significant. All data are presented as mean values+SEM.The presence of PP1γ in human sperm was detected using a polyclonal antibody that recognizes the C-terminal end of the PP1γ1 and PP1γ2 isoforms of human origin, which have a molecular mass of ∼39 kDa . To deteGiven that PP1, PP2B, and PP2A are present in human sperm, we assessed their location by indirect immunofluorescence using the same antibodies as for the western blot assay, except for PP2A, for which we used an antibody obtained from Calbiochem that recognizes the regulatory subunit A, PP2A-A, of 65 kDa . PP1γ waThere are very few studies regarding the function of Ser/Thr PPs during capacitation. To understand their role, we incubated the sperm with different inhibitors and evaluated the appearance of sperm with the B pattern. First, we determined the optimal concentration of phosphatase inhibitors to be used. Sperm were resuspended in capacitating medium (CM) and incubated for 1 h in the presence of different concentrations of inhibitors. The inhibitors were used at the IC50 (as described for other cell types) and at concentrations 10 times lower and 10 times higher. To determine the effect of PP1, we used okadaic acid  ; to evalIn the following experiments, the effect of each inhibitor was tested on sperm resuspended in either non-capacitating medium (NCM)  or in re50 and analyzed immediately  .The most significant finding of the present work is that Ser/Thr PPs play a physiological role during human sperm capacitation. The evidence in support of this claim was obtained by working with specific inhibitors, measuring the phosphatase enzymatic activities, and performing western blots to detect changes in Thr protein phosphorylation. The experimental evidence that supports this assumption is as follows: 1) three members of the Ser/Thr phosphatase families were detected in human sperm, including PP2A; 2) when any of these three PPs were inhibited, the percentage of sperm depicting B pattern increased rapidly and significantly (≤1 min of inhibitor addition); 3) all of the inhibitors used blocked phosphatase enzymatic activity; 4) the enzymatic activity of these three phosphatases decreased during capacitation; 5) phosphorylation of proteins on Thr residues increased during capacitation; and 6) all of the inhibitors used increased Thr protein phosphorylation in a very fast and dramatic manner.Currently, there is extensive knowledge regarding the participation of PKs during capacitation, but information about the presence and participation of PPs during this process is scarce. The fact that PKs have an active participation in the capacitation process leads us to hypothesize that PPs may also have an important role. In particular, it has been reported that human sperm incubated in RCM for less than 1 min showed an immediate greater than twofold increase in PKA activity compared with cells suspended in NCM. This increase in PKA activity was followed by an increase in PKA-dependent phosphorylation that begins within 90 sec The presence of PP2A has not been described in human sperm. In this work, we present evidence for the presence of PP2A. In particular, we show the presence of the catalytic subunit of PP2A, isoforms α and β ; the regulatory subunit A or PR65, isoforms α and β ; and the regulatory subunit B, specifically the PR55 subunit belonging to the B family .Regarding the localization of these PPs, PP1γ appears to be located mainly in the postacrosomal and neck region of human sperm. A less conspicuous label was observed in the middle and principal piece of the flagellum. The subcellular localization of PP1γ has been described in mouse To study the function of these PPs during capacitation, we used a PP2B inhibitor, D, and a PP2A inhibitor, E. We also used OA, which is a potent inhibitor of PP1 and PP2A at different concentrations; PP2A is 100 times more sensitive to OA than PP1 In this regard, we observed two related findings that are noteworthy. First, there was a very rapid increase in sperm exhibiting the B pattern when BSA/HCO3- was added to sperm in NCM (to become RCM); second, there was a very rapid increase in capacitation when the PP1 and PP2B inhibitors were added to sperm incubated either in NCM or RCM. In fact, when BSA/HCO3- or the inhibitors were added to the sperm, the percentage of cells with the B pattern increased significantly and rapidly within 1 min of addition. Shortly thereafter, the percent of capacitated cells reached a plateau. After 2 h of incubation, the percentage of capacitated sperm was similar to that of the sperm incubated in CM without inhibitor.These results are consistent with those reported by Furuya et al. 50 of each inhibitor, and the results strongly suggest the involvement of both PPs during this process.In all of the above-mentioned studies, the concentration of inhibitors used was very high  and that PP2A is inhibited through phosphorylation on Tyr (Tyr307). This effect has been observed in sperm of mouse Finally, we were interested in determining whether the inhibition of these PPs produces changes in the phosphorylation pattern on Thr residues. We first evaluated the pattern of phosphorylation on Thr residues during capacitation in sperm incubated for 1 h in RCM. Naz et al. We also assessed the protein phosphorylation pattern of sperm incubated in RCM in the presence or absence of phosphatase inhibitors. Sperm incubated for short periods of time (≤1 min) in the presence of the inhibitors OA, E, or D exhibit an increase in the phosphorylation of Thr residues of proteins from 42 to 100 kDa. This observation is consistent with the rapid increase in the percentage of capacitated sperm and the rapid decrease in phosphatase activity provoked by the inhibitors. Jha and Shivaji In conclusion, we report evidence that suggests that during capacitation the activity of the Ser/Thr PPs PP1, PP2B and PP2A decreases and that this decrease may be required for successful capacitation in human sperm .Figure S1Effect of high concentrations of okadaic acid on the capacitation of human sperm. Sperm resuspended in CM were treated with different concentrations of okadaic acid. The percent of capacitated cells was evaluated at various times. Results were obtained from five different donors and are expressed as the mean±SEM of the percentage of the B pattern. * p<0.05; *** p<0.001.(TIF)Click here for additional data file.Figure S2Effect of deltamethrin on the capacitation of human sperm. Sperm resuspended in CM were treated with different concentrations of deltamethrin. The percent of capacitated cells was evaluated at various times. Results were obtained from four different donors and are expressed as the mean±SEM of the percentage of the B pattern. *** p<0.001.(TIF)Click here for additional data file.Figure S3Effect of low concentrations of okadaic acid on the capacitation of human sperm. Sperm resuspended in CM were treated with different concentrations of okadaic acid. The percent of capacitated cells was evaluated at various times. Results were obtained from five different donors and are expressed as the mean±SEM of the percentage of the B pattern. * p<0.05; *** p<0.001.(TIF)Click here for additional data file.Figure S4Effects of endothall and dimethylendothall (DME) on the capacitation of human sperm. Sperm resuspended in CM were treated with different concentrations of endothall and with 90 nM DME. The percent of capacitated cells was evaluated at various times. Results were obtained from five different donors and are expressed as the mean±SEM of the percentage of the B pattern. *** p<0.001.(TIF)Click here for additional data file.


The storage proteins from sesame seed, 11S globulin, 7S vicilin and 2S albumin, are official WHO/IUIS allergens. However, little is known about the IgE reactivity to the individual sesame allergen components.To investigate the IgE reactivity to purified sesame storage proteins in sera from sesame allergic Japanese children.® matrix and quantitative IgE responses in sera from five sesame allergic Japanese children were measured.The three storage proteins were all purified from unpolished sesame seeds extracted at pH 7,5. The 11S globulin and the 7S vicilin were enriched by precipitation in water. Pure 11S globulin was obtained by a further precipitation with 35% ammonium sulfate followed by anion exchange chromatography. Pure 7S vicilin was obtained by adding gel filtration and anion exchange chromatography. The 2S albumin was purified by cation exchange chromatography and gel filtration. Identities were confirmed by MALDI-TOF analysis. The three purified storage proteins were conjugated to the ImmunoCAPAll the sera from sesame allergic Japanese children showed specific IgE reactivity to sesame extract. Out of the five sera, four had IgE response to 11S globulin, all five to 7S vicilin and three to 2S albumin.IgE reactivity to all three storage proteins in sesame seed, 11S globulin, 7S vicilin and 2S albumin, was shown in sera from the sesame allergic Japanese children.


In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4+ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.We previously reported that protein kinase D2 (PKD2) in T cells is promptly activated after T-cell receptor (TCR) stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A). In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity.  For example, SET protein together with another nuclear phosphoprotein pp32 forms a subunit of inhibitor for histone acetyltransferase complex with “histone masking” activity, and binding of these proteins to histones prevents their acetylation by transcriptional coactivators The phosphoprotein SET  is a member of PKD family protein serine/threonine kinases and expressed in various cell types et al. also showed that the kinase activity of PKD2 was important for effecter cytokine production after TCR engagement C. elegans lacking DKF-2, an ortholog of mammalian PKD, was more susceptible for pathogenic bacterial infection and DKF-2 was shown to be positively involved in innate immunity in C. elegansOver-expression of PKD2 and TCR-stimulation up-regulated interleukin-2 promoter activity in Jurkat cells Whereas those observations suggest the positive role of PKDs in immune responses, it was shown that PKD2 was responsible for the interferon α-induced phosphorylation of a degron of the α interferon receptor and therefore for its accelerated degradation by ubiquitination, which could neutralize the defensive functions of type I IFN In the present work, we identified one of the phosphorylation sites of SET protein by PKD2 and potential role of SET phosphorylation by PKD2 is discussed.2 fragment was from PIERCE Biotechnology . Myelin basic protein , anti-PP2A C-subunit antibody and purified PP2A AC heterodimer were obtained from Upstate Biotechnology . Gö6976 and Gö6983 were from Calbiochem . Jurkat cell nuclear extract was purchased from Active Motif (Carlsbad CA). Poly-L-Lysine Agarose and phytohemagglutinin (PHA) were form Sigma-Aldrich .Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection . Jurkat cells expressing GFP-tagged constitutively active (CA) mutant and GFP-tagged kinase dead (KD) mutant of PKD2  were prepared as reported previously Full-length human SETβ cDNA was obtained from total RNA of Jurkat cell using RT-PCR and inserted into a pET28a vector  for preparation of recombinant proteins or a pAcGFP1-C1 vector for Jurkat cell-transfection. Mutations of SET were introduced by overlapping PCR.2 fragment (8 µg/ml) and stimulated for 1 h at 37°C, or stimulated with PHA (2 µg/ml) at 37°C for 12 hrs. Human CD4+ T cell clone was stimulated by co-culturing with mouse L cells expressing its cognate ligand Jurkat cells were stimulated with anti-CD3ε mAb (2 µg/ml) for 10 min followed by addition of anti-mouse IgG F(ab′)Jurkat cells expressing GFP-SET-WT were stimulated by PHA and lysed using a lysis buffer supplied with a PhosphoProtein Purification Kit (Qiagen) according to the manufacturer's instruction. In some experiments, cells were treated under the presence of PKD2 inhibitor Gö6976 (3 µM) before and during the PHA stimulation or the cells were treated with siRNA as described below before the stimulation. The fluorescence of GFP in the lysate was measured at excitation 475 nm and emission 505 nm using a Hitachi Fluorescence Spectrometer F-2500 . Each lysate containing the same amount of fluorescence was applied to the PhosphoProtein Purification Columns (Qiagen). Phospho-GFP-SET-WT recovered was subjected to SDS-PAGE and transferred onto a PVDF membrane. The phospho-GFP-SET-WT was detected and quantified by anti-GFP antibody and horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescent detection .7) treated with anti-CD3ε mAb and anti-mouse IgG F(ab′)2 fragment were lysed and immunoprecipitated with anti-PKD2 antibody as described previously 2 and 1 mM DTT) containing 16 µg of heat-inactivated  Jurkat nuclear extract (Active Motif) and either 0.25 MBq [γ-32P]-ATP or 25 µM cold ATP at 30°C for 10 min. The kinase reaction with [γ-32P]-ATP was performed in the presence of 3 µM of Gö6983 or Gö6976. Both kinase reaction mixtures with hot and cold ATPs were submitted to isoelectric focusing on pH 3–10 non-linear immobilized pH gradient strips  followed by 11% second dimension SDS- PAGE in parallel. After the gels were stained with syproruby reagent (Bio-Rad), the gels with radioactivity were sandwiched with clear cellophane sheets, dried and exposed to X-ray films . UV-illuminated dried gel sheets were overlaid onto the developed X-ray films to identify radioactive protein spots. The corresponding syproruby-stained protein spots on the cold gels were excised and subjected to trypsin-digestion and mass-spectrometric analysis.Jurkat cells (1×10E. coli strain (Novagen). The 6×His-tagged SET protein expression was induced according to the manufacturer's instruction. The bacterial pellet was sonicated and the soluble recombinant SET proteins were fractionated by poly-L-lysine-agarose beads column according to the method described by Li et al.pET28a-SET plasmids were introduced into BL21(DE3) 32P-labeled by recombinant PKA (New England BioLabs) according to the manufacturer's instruction. 32P-MBP, recombinant SET proteins and purified PP2A (Upstate) were diluted with phosphatase buffer . After mixing 16 µl of indicated concentrations of the recombinant SET and 2 µl PP2A (37 µU) and incubating at 4°C for 15 min, 2 µl 32P-MBP was added and left at 37°C for 10 min. The reaction was stopped by adding 6 µl of sample buffer and 20 µl of the reaction mixture was subjected to 15% SDS-PAGE. The gels were dried and the remaining radioactivity of MBP was quantified by BAS-2000 image analyzer (Fujifilm). In ex vivo experiments, the PP2A phosphatase activity from Jurkat or human CD4+ T cell clone were estimated by the phosphorylation status at Tyr307 of the catalytic subunit of PP2A by western blotting using anti-PP2A C-subunit phospho-Tyr307 Dephosphorylated MBP was PKD2 were designed as follows: siRNA1 sense, 5′-GACAAACUGCGCUUCCCUACC-3′ and antisense, 5′-UAGGGAAGCGCAGUUUGUCAA-3′; siRNA2 sense, 5′- GUUCGAGACGCCUGAGAAAGU-3′ and antisense, 5′- UUUCUCAGGCGUCUCGAACAU-3′. Three µl of each duplex siRNA (100 µM) was added to Jurkat cell suspension (1.5×107/100 µl) and electroporated with a Nucleofector II (Lonza) according to the manufacturer's instructions. The siRNA1 had no effect on PKD2 expression level and used as a negative control.siRNAs for human 6) or human CD4+ T cell clone (1.5×106) were lyzed with 15 µl of the lysis buffer  and the supernatant was subjected to SDS-PAGE. The proteins were electro-blotted onto a PVDF membrane. PKD2, SET, GFP-tagged recombinant SET proteins, ERK1, phospho-ERK and β-actin were probed with respective antibodies and horseradish peroxidase-conjugated secondary antibody followed by the ECL detection. A lumino image analyzer  was used to quantify the chemiluminescence.Jurkat cells , showing the back ground kinase activity in the nuclear extract was well inactivated by mild heat inactivation. As shown in i, Applied Biosystems, Foster City, CA). The MS/MS analysis  applied with lysate of Jurkat cells over-expressing constitutively active PKD2 Since activated PKD2 translocates into the nucleus r Gö6983  that doer Gö6983 . The thir Gö6983 . Incorpoanalysis  suggestein vivo, Jurkat cells expressing GFP-SET-WT were stimulated with PHA and phospho-GFP-SET-WT (GFP-pSET-WT) was recovered and quantified by western blot using anti-GFP antibody as described in the Method section. The amount of GFP-pSET-WT was up-regulated after stimulation  were stimulated with PHA in the absence or presence of PKD2 inhibitor Gö6976 and the amount of phospho-GFP-SET-S171A recovered from each cell lysate was compared by the western blotting using anti-GFP antibody. Contrary to the data shown in To examine whether Ser171 is involved in SET phosphorylation 32P-MBP was used as a substrate. As shown in Since SET protein is known to be a natural inhibitor for PP2A in vivo. To ask this possibility, the relationship between PKD2 and activity of PP2A in ex vivo system was investigated using a TCR stimulated human CD4+ T cell clone or Jurkat cells. Since it is reported that phosphorylation status of Tyr307 of PP2A catalytic subunit inversely correlates with the activity of PP2A The data presented above suggested the possibility that PKD2 may regulate PP2A activity through SET phosphorylation + T cell clone was stimulated by co-culturing with mouse L cells expressing its cognate TCR-ligand, HLA-DR4 covalently-linked with the antigenic peptide + T cell clone without inhibitor  fits to the phosphorylation motif of PKD1 (LxR/KxxS) Since the all Ser/Thr to Ala mutant of C-terminal half of 6×His-tag SET protein was not phosphorylated by PKD2, Ser/Thr residues present in the linker portion between 6×His-tag and SET were not target phosohorylation sites for PKD2. This suggested that there must be other target phosohorylation sites of PKD2 in the N-terminal half of SET protein since it also incorporated the in vivo32P –labeling of SET, ex vivo detection of phosphorylated SET by anti-phospho-Ser antibody or by the Phos-tag detection kit , the clear difference of the phosphorylation status of SET before and after stimulation was not observed at this moment.The interaction between SET and PKD2 was shown in Jurkat cells since the anti-PKD2 antibody co-immunoprecipitated SET from the Jurkat cell lysate . Howeverin vitroSET is a multi-functional protein and has an acidic domain rich in Glu and Asp residues at its very C-terminus (amino acid positions 224–277). The acidic domain is indispensable for its inhibitory effect on the histone acetyltransferases in vitro phosphatase assay using immunoprecipitated PP2A from activated T cells by colorimetric assays using p-nitrophenol or Malachite Green + cells by over-expressing SET protein The in vivo, overexpression of non-phosohorylation-mimic mutant of SET in TCR-stimulated Jurkat cells resulted in the down-regulation of ERK phosphorylation as well as PP2A activity, suggesting the involvement of phosphorylation status of SET and PP2A activity in the ERK activation. Together, it is tempting to hypothesize that PKD2 is a positive regulator of T cell activation through SET phosphorylation and up-regulation of PP2A activity, which results in the activation of MAPK pathway. This notion is in accordant with our first finding that partially-agonistic TCR ligands induced ZAP-70 independent T cell proliferation and interferon-γ production, which was completely suppressed by PKD2 inhibitor There are many reports providing the evidences that PP2A dephosphorylates the inhibitory phosphate at Ser259 of Raf-1, and thus PP2A activates Raf-1 and then activates ERK-dependent signaling pathway In conclusion, the roles of PKD2 and its substrate SET in T cell activation were investigated and we found that PKD2 phosphorylates Ser171 of SET, which resulted in the reduction of its inhibitory effect on PP2A phosphatase activity.Supporting Information S1Co-immunoprecipitation of SET by anti-PKD2 antibody from Jurkat cells lysate. Jurkat cells (1×107) and GFP-SET-WT expressing Jurkat cells (1×107) were stimulated with PMA (20 ng/ml) and ionomycin (2 µg/ml) for 30 min at 37°C and lysed. Each cell lysate was immunoprecipitated with anti-PKD2, anti-SET or anti-GFP antibodies and Protein A beads. The anti-PKD2 antibody co-immunoprecipitated SET (left pannels) from Jurkat cell lysate. Reciprocally, the anti-SET antibody failed to co-immunoprecipitate PKD2 from Jurkat cell lysate (middle panels), while the anti-GFP antibody could co-immunoprecipitate PKD2 (right pannels) with GFP-SET.(TIF)Click here for additional data file.Supporting Information S2PP2A phosphatase activity was up-regulated after TCR stimulation in Jurkat cells. Jurkat cells (1×106) were left untreated or stimurated with anti-CD3ε antibody and anti-mouse IgG antibody for 30 min. Each cell lysate was immunoprecipitated with anti-PP2A C-subunit antibody (anti-PP2A) or non-specific control antibody (IgG) and Protein A beads. The immunocomplex were incubated with 32P-MBP as a substrate. The reaction mixture was separated with SDS-PAGE. The autoradiogram was shown in the upper panel. The phosphatase activity was measured as a remaining radioactivity of 32P-MBP. The radioactivity of 32P-MBP treated with the immune complex precipitated with non-specific control antibody was assigned to be 1 (lower panel). Student's t test analysis for the PP2A activity of stimulated versus non-stimulated was done. *, p<0.01 (n = 4).(TIF)Click here for additional data file.