COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called   •Alternative splicing of noncoding antisense transcripts affects flowering•Arabidopsis FLC gene transcription modulated by lncRNA isoforms•Positive feedback links chromatin state and antisense transcript splicing•Quantitative gene regulation by coupling antisense splicing to chromatin states Arabidopsis floral repressor gene FLC are alternatively spliced. Interfering with this alternative splicing alters FLC transcription quantitatively and affects the timing of flowering. Thus, this work reveals a function for a long noncoding antisense transcript in Arabidopsis.Marquardt et al. demonstrate that noncoding transcripts antisense to the  The biological significance of non-protein-coding genomic sequences has been an issue for decades . This haSeveral in-depth studies in yeast have shown that noncoding transcripts have the potential to regulate gene expression through transcriptional interference or recruitment of chromatin modifiers . HoweverArabidopsis FLOWERING LOCUS C (FLC) gene, a developmental regulator that controls the timing of the switch to reproductive development. FLC encodes a MADS box transcriptional regulator that represses flowering, and FLC expression quantitatively correlates with flowering time  site, increasing histone methylation in the gene body and upregulating FLC transcription. We also show a positive feedback mechanism between gene body histone methylation and COOLAIR processing. The involvement of COOLAIR splicing in this mechanism was supported through both disruption of COOLAIR production and cis mutation of the antisense proximal splice acceptor site. Cotranscriptional coupling mechanisms such as this may be of widespread importance in the quantitative regulation of gene expression.We have continued to investigate the transcriptional circuitry at FLC repression by FCA  to enhance FCA activity and establish low levels of FLC, a FLC-LUCIFERASE (FLC-LUC) reporter to efficiently monitor FLC levels, and a functional FRIGIDA (FRI) allele to amplify changes in FLC expression to increase sensitivity of detection (Arabidopsis accessions such as Landsberg erecta (Ler) and Columbia (Col) contain loss-of-function fri alleles, and the functional FRI we added originated from a Swedish accession  81   81 B. sof81 as a sof , and wasas a sof B. The muenotype) . At1g800iceosome . The sofon 1,891 C within on 1,891 D. The muon 1,891 C. The RNon 1,891  that preon 1,891 .PRP8 (sus2) plants are embryonic lethal, indicating the mutation in sof81 (referred to from now as prp8-6) is hypomorphic. The prp8-6 mutant phenotype was rescued by a genomic PRP8 clone  and one copy of the prp8-6 allele showed no complementation based on FLC-LUC bioluminescence and flowering-time analyses  transcribed at low levels , including the presence of a Mutator transposon at the 3′ end of intron 1 -LUC in prp8-6, both of which were increased compared to the progenitor  and delayed flowering much more extensively when the 35S-FCAγ transgene was crossed out, but was epistatic (nonadditive) with the loss-of-function mutation of FCA, fca-1. Consistent with this, when prp8-6 was combined with just FRI, the expression of FLC was significantly higher  indicated no effect of prp8-6 on these splicing events  site choice in the COOLAIR transcript. Alternative poly(A) sites clustered in proximal and distal regions have been characterized  site and promoted use of the distal site . This generated an FLC transgene (named FLCtex) that encoded a sense transcript with a different 3′ UTR and lacked the COOLAIR promoter, exon 1, and intron 1. The FLCtex and wild-type FLC constructs were transformed into a loss-of-function FLC genotype (FRI flc-2), which has a deletion/rearrangement within the endogenous FLC gene (FLC transcription (assayed as spliced transcript accumulation) was ∼3-fold higher in FLCtex lines compared to FLC transgenic lines  site disrupted transcriptional repression of FLC  site, might be an important factor in the increased expression of FLC in prp8. We therefore specifically blocked splicing of this intron by site-directed mutagenesis of the terminal intronic dinucleotide AG to AA  were generated in a FRI flc-2 genotype, each with and without the prp8-6 mutation, and analyzed in pools. The AG-to-AA mutation (COOLAIRAA) significantly reduced splicing efficiency of the intron and increased levels of FLC expression  site usage.Since reduced use of the n of FLC , we reasAG to AA A. Mutatipression B. When c-6 alone B, suggesg events A. Proxim changes B. OveralCOOLAIR transcripts has been shown to trigger changes in histone methylation, increased transcription as assayed by unspliced transcript production, and RNA Pol II occupancy at the FLC locus  site  site A and S6Butations . We addrpression A. As witCOOLAIR might be coupled with the chromatin state at FLC in two ways. First, we analyzed COOLAIR splicing and polyadenylation in the fld demethylase mutant. The splicing efficiency of antisense introns class Ii was significantly reduced in fld, as in fca  site choice  is a heritable human disease that describes progressive degeneration of the retina during development, leading to blindness; one of the heterogeneous causes of RP is a set of mutations all clustering to the C terminus of PRP8  site. Autonomous pathway mutations may be useful in the dissection of the tight connection between poly(A) site choice and last intron acceptor site choice  (The  of PRP8 . While te choice . Read-thmutants) ; howeverCOOLAIR and FLC transcription. Analysis of fld, the histone K4 demethylase mutant  that was available through the John Innes Genome Centre. A 10 kb genomic PRP8 region was amplified by PCR with the oligonucleotides PRP8-SacII-SbfI-F and PRP8-KpnI-R using pJATY50P17 as template with Phusion DNA polymerase (NEB). The PCR fragment was cloned into the binary plant transformation vector pCambia-1300, conferring hygromycin resistance in plants via SbfI/KpnI cloning to generate ASM4. The cloned genomic PRP8 region in ASM4 was sequenced to verify the absence of mutations. ASM4 was transformed into sof81 mutants by Agrobacterium mediated floral-dip transformation, and hygromycin-resistant T1 transformants were isolated (n > 10). The activity of the FLC-LUC reporter of the transformants was compared to untransformed sof81 mutant controls.The genomic region encompassing the FLC was cloned as a genomic SacI fragment (∼12 kb) into the Arabidopsis binary vector pCambia-1300, which confers hygromycin resistance in plants. To generate COOLAIRAA, fragments F1  and F2 (311 bp) were amplified from FLC with primers for F1 (FLC3ss_F1-forward and FLC3ss_F1-reverse) and F2 (FLC3ss_F2-forward and FLC3ss_F2- reverse) containing a mutated sequence for the 3′ splice site of FLC antisense class Ii intron (AA instead of AG). PCR amplification was performed with Phusion polymerase (NEB). Resulting fragments F1 and F2 with overlapping ends were fused together in 1:1 molar ratio by PCR amplification with Phusion polymerase (NEB) employing the forward primer for F1 and the reverse primer for F2. The resulting fragment was digested with NheI and BglII, gel purified, and subsequently cloned into an SphI fragment of FLC, replacing the wild-type NheI-BglII fragment. The resulting SphI fragment with the mutated class Ii antisense 3′ splice site was inserted into FLC-pCambia-1300. This mutation creates a recognition site for DraI (TTTAAA), which has been used for genotyping the hygromycin resistant transformants to verify presence of the COOLAIRAA mutation.prp8-6/flc-2/FRI and PRP8/flc-2/FRI were obtained from crosses of prp8-6 and flc-2/FRI. The F2 homozygotes were transformed using Agrobacterium-mediated transformation of floral buds with the either FLC-pCambia-1300 or COOLAIRAA-pCambia-1300. The seeds from a total of 49 T1 (first generation) transformants  were sown on GM medium without glucose and selected for hygromycin resistance (T2 generation). RNA for analysis was extracted from 4-week old seedlings.F2 homozygotes of the following genotypes: COOLAIRTEX, the sequence TAGCCACC that contains FLC translational stop TAG codon was mutagenized to create EheI restriction site TGGCGCCC. A SspI-SspI fragment containing the strong RBCS terminator (706 bp) was PCR amplified and cloned in sense direction between EheI and SwaI restriction sites .For cloning FLCtex seeds were collected from four homozygous plants of FLCtex/flc-2/prp8-6/FRI and five homozygous plants of FLCtex/flc-2/PRP8/FRI. These plants were obtained from the three independent crosses of FLCtex/flc-2/FRI to prp8-6/Ler. As a control for the FLCtex analysis, the flc-2/FRI plants were transformed with pSLJ-FLC15 (10 kg clone of Columbia FLC gene) and crossed with prp8-6/Ler (two independent crosses). Three plants from either FLC/flc-2/prp8-6/FRI or FLC/flc-2/PRP8/FRI were obtained. The seedlings from FLCtex and corresponding pSLJ-FLC transgenic plants were grown on GM medium without glucose and BASTA resistant transformants were isolated for analysis.To analyze the effect of FLC mRNA analysis, reverse transcription was performed using FLC specific reverse primers with SuperScript®III Reverse Transcriptase (Invitrogen). qPCR analysis was performed on LightCycler480®II (ROCHE) with primers FLC Unspliced_LP and FLC Unspliced_RP for the unspliced sense FLC transcript and with primers FLC Spliced_LP and FLC Spliced_RP for the spliced sense FLC transcript. qPCR data was normalized to UBC (which was amplified with primers UBC-F and UBC-R). The primers are described in For the sense To measure the splicing efficiency of class Ii intron, 5 μg of total RNA isolated from seedlings were reverse-transcribed into cDNA, primed by Int1_RT, which is located in the exon 2 of class I and class II ii,  for proximal poly(A) site transcript oligo(dT) primer was used for the reverse transcription and forward primer, set1_RP, and reverse primer, LP_FLCin6polyA, used for the qPCR analysis . The primers are summarized in the The following primers were employed for the analysis of the analysis , and (b)


In this study, we hypothesized that androgen-deprivation therapy (ADT) in prostate cancer, although initially efficient, induces changes in the tumor kinome, which subsequently promote development of castration-resistant (CR) disease. Recognizing the correlation between tumor hypoxia and poor prognosis in prostate cancer, we further hypothesized that such changes might be influenced by hypoxia. Microarrays with 144 kinase peptide substrates were applied to analyze CWR22 prostate carcinoma xenograft samples from ADT-naïve, androgen-deprived (AD), long-term AD (ADL), and CR disease stages. The impact of hypoxia was assessed by matching the xenograft kinase activity profiles with those acquired from hypoxic and normoxic prostate carcinoma cell cultures, whereas the clinical relevance was evaluated by analyzing prostatectomy tumor samples from patients with locally advanced disease, either in ADT-naïve or early CR disease stages. By using this novel peptide substrate microarray method we revealed high kinase activity mediated by signal transducer and activator of transcription 5A (STAT5A) in CR prostate cancer. Additionally, we uncovered high STAT5A kinase activity already in regressing ADL xenografts, before renewed CR growth was evidenced. Finally, since increased STAT5A kinase activity also was detected after exposing prostate carcinoma cells to hypoxia, we propose long-term ADT to induce tumor hypoxia and stimulate STAT5A kinase activity, subsequently leading to renewed CR tumor growth. Hence, the study detected STAT5A as a candidate to be further investigated for its potential as marker of advanced prostate cancer and as possible therapeutic target protein. The initial growth of a malignant prostate tumor is stimulated by androgens In vitro studies have shown that hypoxia increases the activity and sensitivity of the androgen receptor Hypoxia is a common microenvironmental factor of solid tumors, promoting tumor growth as well as angiogenesis, metastasis, and therapy resistance It has for many years been well known that disease progression and development of treatment resistance in cancer generally are characterized by altered expression and activity of key mediators in cellular signal transduction, regulating processes such as proliferation, invasion, and angiogenesis in vivo preclinical prostate cancer model reflecting different stages in development of CR disease. The impact of hypoxia was assessed by matching this model’s kinase activity profiles with those generated from hypoxic and normoxic prostate carcinoma cell cultures. Clinical relevance was evaluated by analyzing prostatectomy tumor samples from patients with locally advanced disease, either in ADT-naïve or early CR disease stages.We hypothesized that ADT in prostate cancer, although initially efficient, induces changes in the activity of the tumor kinome that subsequently lead to renewed, CR tumor growth. To examine this hypothesis, microarrays with kinase peptide substrates, mainly representing tyrosine residues, were applied to analyze samples from an All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee at Department of Comparative Medicine, Oslo University Hospital (Permit Number: 32-1984), in compliance with guidelines on animal welfare of the Norwegian National Committee for Animal Experiments. The clinical study protocol was approved by the Regional Committee for Medical and Health Research Ethics  and was in accordance with the Helsinki Declaration. Written informed consent was required for participation.3) of the human, androgen-sensitive CWR22 prostate carcinoma xenograft Tissue fragments , 3.8 mg/ml xylazine , and 0.1 mg/ml butorphanol ), diluted 1∶5 in sterile water, in a dose of 7.5 µl/g. Analgesia was given to castrated animals as s.c. injections of buprenorphine , in a dose of 0.1 mg/kg.Blood samples for prostate-specific antigen (PSA) analysis were withdrawn from all animals’ femoral veins right before euthanasia. The samples were allowed to coagulate before being centrifuged and stored at −80°C until all samples were analyzed simultaneously. Free and total serum PSA levels were assayed by the fluoroimmunonometric AutoDELFIA ProStatus™ PSA Free/Total kit . Euthanasia of animals was performed by cervical dislocations and immediately followed by excision of tumor tissue, which directly was snap-frozen in liquid nitrogen, and stored at −80°C.For the purpose of validating preclinical data, four prostate cancer patients that had undergone radical prostatectomy with clear resection margins were included in the study . The pat3 was cut of all xenograft samples, the required number of sections to cut calculated after measuring the samples’ width and length. By identical procedures, ∼5 mm3 was sectioned of each prostatectomy biopsy  specimen.Xenografts and surgical biopsies from the prostatectomy specimens, fresh-frozen immediately following resection, were first evaluated for tumor cell content, after being sectioned using a cryostat microtome and stained with hematoxylin and eosin. Any normal tissue present in tumor samples was roughly removed by dissection guided by the stains. All tumor specimens used had tumor cell content higher than 80%. The specimens were subsequently cut in 10 µm sections, continuously kept frozen and immediately re-stored at −80°C. A volume of ∼3 mmProcedures for preparation of protein lysates and generation of kinase activity profiles are described in 2-transformed, before the mean replicate signal intensity within each experiment was calculated for each peptide substrate. To correct for possible experiment-to-experiment variation, the mean of the four experimental groups’ signal intensities for each peptide substrate was subtracted from each group’s peptide substrate signal intensity. The microarray data are submitted to ArrayExpress (http://www.ebi.ac.uk/arrayexpress/); accession number E-MTAB-1572.After visual check and background subtraction, endpoint signal intensities  for each spot, i.e. for each kinase peptide substrate per array, were calculated by BioNavigator . A small number of negative signal intensities were managed by subtracting the 1% quantile of all data and setting remaining signal intensities less than 1 to 1. Subsequently, all signal intensities were loghttp://uniprot.org; May 15, 2012) and HUGO Gene Nomenclature Committee (HGNC)  Statistical analysis was subsequently performed in PASW Statistics 18.0 , with p<0.05 considered as statistically significant. ANOVA was applied to identify peptide substrates with significantly different phosphorylation levels between the experimental groups . The Dunnett’s many-to-one  post-hoc test adjusted for multiple comparisons. The peptide substrates’ protein and gene names, including information about the cellular processes they are involved in, were retrieved using UniProt  were analyzed from each patient, on one Tyrosine PamChip®96 plate, at the PamGene International B.V. location in ‘s-Hertogenbosch, The Netherlands. Sample running was performed as previously described 2 flasks at 37°C in 95% air/5% CO2, and subcultured twice weekly to maintain exponential growth.The 22Rv1, PC3, and DU145 human prostate carcinoma cell lines were obtained from the American Type Culture Collection (ATCC). All cell lines were authenticated by short tandem repeat (STR) profiling based on 16 polymorphic markers in use by ATCC and paternity testing, and confirmed to be mycoplasma-free . All cell lines were maintained in RPMI 1640 medium  supplemented with 10% fetal calf serum, 1% L-Glutamine , and 1% penicillin/streptomycin (Invitrogen). The cells were routinely grown as a monolayer in 75 cm2) was achieved by 5% CO2 and residual N2. Severe hypoxia (anoxia) was obtained by 5% CO2 and a mixture of 10% H2 and 90% N2 in combination with a catalyst that catalyzed the conversion of H2 with any O2 left to H2O. All normoxia and hypoxia experiments were performed thrice in order to generate three biological replicates of all experimental conditions.To investigate changes in kinase activity induced by hypoxia, exponentially growing cells at ∼60% confluence, with fresh medium, were transferred to a hypoxia chamber for 24 hours . Moderate hypoxia (1% OPreparation of protein lysates is described in 2) (Sigma Aldrich) for 4 hours before protein lysates were made. Treatment with CoCl2 induces HIF-1α expression by binding to the PAS domain, which blocks the HIF-1α pVHL binding, thereby giving HIF-1α stabilization To simulate hypoxic exposure and expression of the hypoxia-inducible factor-1α (HIF-1α) cells were exposed to 100 µM of the hypoxia-mimetic agent cobalt chloride , anti-STAT5A (Sigma-Aldrich), and anti-phospho STAT5A/B (Y694/Y699) . Mouse monoclonal anti-γ-tubulin (Sigma-Aldrich) was used as loading control. Secondary antibodies were peroxidase-conjugated goat anti-rabbit and donkey anti-mouse . Proteins were visualized by chemiluminescence using SuperSignal West Dura Extended Duration Substrate  or LumiGLO Chemiluminescent Substrate .The CWR22 prostate carcinoma xenografts exhibited volume response patterns following castration similar to those seen in patients, and developed CR xenografts after 5.4±0.4 months . When acFrom analysis of the kinase activity profiles generated by the xenografts, phosphorylation of 35 of the kinase peptide substrates was found to be significantly affected (reduced or increased) after ADT by castration . These s2 ratio and s.e.m. for all 35 significant substrates are shown for the three treatment groups versus ADT-naïve xenografts, with blue color indicating negative log2 ratio and red color indicating positive log2 ratio, respectively. In 10 p-values versus log2 ratios) are visualizing the kinase peptide substrates with the most significant and/or highest log2 ratios between treated and ADT-naïve tumors’ phosphorylation levels.Compared to ADT-naïve xenografts, some substrate phosphorylation levels generated by AD xenografts were weakly decreased, while others were increased. In 2 ratio versus ADT-naïve xenografts of 1.5±0.3 for STAT1 (p = 0.004) and 1.5±0.7 for STAT5A (p = 0.019). Phosphorylation by CR xenografts was for the majority of the substrates restored at the same levels as for ADT-naïve xenografts. However, also from the CR xenografts, the phosphorylation of the STAT1 and STAT5A substrates was high, for STAT5A being significant compared to ADT-naïve xenografts .In accordance with the significant tumor growth inhibition following long-term ADT the level of substrate phosphorylation by ADL xenografts was strongly decreased for 32 of the 35 kinase peptide substrates, compared to the respective phosphorylation levels originating from exponentially growing ADT-naïve xenografts. Two of the three remaining kinase peptide substrates, representing the signal transducer and activator of transcription 1 and 5A (STAT1 and STAT5A), demonstrated significantly increased phosphorylation by ADL xenografts, with a logTo assess clinical relevance, kinase activity profiles of prostatectomy samples from prostate cancer patients were generated. A kinase activity signature was retrieved from the 100 kinase peptide substrates with highest phosphorylation levels, for xenografts and patients’ tumor samples. For xenografts, the signature was represented by the mean of all ADT-naïve xenografts. For clinical samples, the signature was expressed by the mean of the three ADT-naïve tumor samples , it was substantially higher in the early CR patient; log2 ratio = 2.6 (n = 1)  .From the results achieved in xenografts and prostatectomies, combined with the recognition of the potential role of hypoxia in development of CR disease 2) and severe (<0.02% O2) hypoxia for 24 hours. The 22Rv1 cell line is androgen-responsive and originally derived from the relapsed CWR22 xenograft 2 ratios compared to normoxic cells were 0.47±0.27 (p>0.05) for the moderately hypoxic cells and 0.63±0.31 (p = 0.020) for the severely hypoxic cells. A list of the 22 kinase peptide substrates significantly affected by hypoxia is provided in To further inspect this hypothesis, kinase activity profiles were generated from normoxic 22Rv1 prostate carcinoma cells, as well as from cells exposed to moderate (1% OA Western immunoblot correspondingly confirmed an increased expression level of phospho-STAT5A/B (Y694/699) in severely hypoxic 22Rv1 cells, compared to normoxic 22Rv1 cells . The exp2, a chemical inducer of HIF-1α. As seen in 2 induced increased expression of both HIF-1α and phospho-STAT5A/B in all cell lines. The STAT5A protein expression was increased in one cell line and unchanged in the two other cell lines. Since HIF-1α specifically induced an increase in STAT5 phosphorylation these results supports that hypoxia is the cause of increased STAT5 activity, through a HIF-1α-mediated mechanism.To elucidate whether STAT5A activation was directly induced by HIF-1α activation we treated the cells with CoClThe current study identified increased phosphorylation level of the STAT5A peptide substrate by ADL and CR prostate carcinoma xenografts, compared to by ADT-naïve xenografts. Similarly, in a limited population of prostate cancer patients studied, low phosphorylation levels of the STAT5A substrate by tumors from ADT-naïve patients’ were revealed, whereas the STAT5A phosphorylation level generated by the tumor from an early CR disease case was substantially higher. Thus, an association between high kinase activity mediated by STAT5A and development of CR disease was uncovered both in xenografts and patient tumor samples. Further, we detected higher phosphorylation levels of the STAT5A substrate by hypoxic prostate carcinoma cells than by the normoxic counterpart. Hypoxia-mediated activation of STAT5A signaling has to our knowledge not been reported in prostate cancer.STAT5 belongs to the STAT gene family of transcription factors in vitroin vivo preclinical model Previous studies have identified STAT5A/B to be a critical survival factor in prostate cancer cells in vitro model In liver cancer cells, hypoxia-induced activation of STAT5B was shown to regulate expression of insulin-like growth factors Hence, several studies propose a captivating link between the STAT proteins and survival of tumor endothelium during angiogenesis. Interestingly, in our study, several substrates representing kinases involved in angiogenesis were reactivated, i.e. presenting significantly higher level of substrate phosphorylation by CR xenografts compared to by ADL xenografts. These included PECAM1, several VEGFRs, PDGFRs, PIK3R1, and EPOR . SignaliIn summary, by using a novel peptide substrate microarray method, we were able to generate kinase activity profiles of preclinical and clinical samples from different stages in development of CR prostate cancer. From these profiles, previously published observations of high STAT5A activity in CR prostate cancer were confirmed. In addition, we also detected high STAT5A kinase activity already in regressing xenografts after long-term ADT. Since increased STAT5A kinase activity was generated by exposing prostate carcinoma cells to hypoxia, we propose that long-term ADT may induce tumor hypoxia and stimulate STAT5A kinase activity, which subsequently may lead to renewed, CR tumor growth. Hence, the study detected STAT5A as a candidate to be further investigated for its potential as marker of advanced prostate cancer and as possible therapeutic target protein.Text S1Materials and methods.(DOC)Click here for additional data file.Text S2Evaluation of interchip and intrachip variation.(DOC)Click here for additional data file.


Francisella tularensis is an infectious, gram-negative, intracellular microorganism, and the cause of tularemia. Invasion of host cells by intracellular pathogens like Francisella is initiated by their interaction with different host cell membrane receptors and the rapid phosphorylation of different downstream signaling molecules. PI3K and Syk have been shown to be involved in F. tularensis host cell entry, and both of these signaling molecules are associated with the master regulator serine/threonine kinase mTOR; yet the involvement of mTOR in F. tularensis invasion of host cells has not been assessed. Here, we report that infection of macrophages with F. tularensis triggers the phosphorylation of mTOR downstream effector molecules, and that signaling via TLR2 is necessary for these events. Inhibition of mTOR or of PI3K, ERK, or p38, but not Akt signaling, downregulates the levels of phosphorylation of mTOR downstream targets, and significantly reduces the number of F. tularensis cells invading macrophages. Moreover, while phosphorylation of mTOR downstream effectors occurs via the PI3K pathway, it also involves PLCγ1 and Ca2+ signaling. Furthermore, abrogation of PLC or Ca2+ signaling revealed their important role in the ability of F. tularensis to invade host cells. Together, these findings suggest that F. tularensis invasion of primary macrophages utilize a myriad of host signaling pathways to ensure effective cell entry. Francisella tularensis subspecies tularensis (Type A) and subspecies holartica (Type B) are highly infectious, Gram-negative, intracellular pathogens that cause tularemia, a disease with significant morbidity and mortality in humans and other mammals. Due to its ease of infection and means of dissemination, these F. tularensis subspecies are classified as select agents  for the indicated time periods. Macrophages derived from KO mice were stimulated with F. tularensis LVS for the indicated times. Cells were then washed with PBS and lysed as previously described [TM Protein Assay Kit . Equivalent amounts of protein from cell lysates were separated by SDS-PAGE on a 7.5 or 10% Tris–HCl gel . Protein was electrotransferred to immobilon-P transfer membranes  and Western analysis was carried out with specific antibodies against the total and/or phosphorylated forms of extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), p38 mitogen-activated protein kinase (Thr180/Tyr182), Akt (Ser473), PI3K p85 (Tyr458/p55), p70S6K (Thr389) and (Thr421/Ser424), 4E-BP1 (Ser65), eI-F4E (Ser209), S6 (Ser235/236) and (Ser240/244) and rictor . Detection of antibodies was carried out using horseradish peroxidase-linked rabbit or mouse anti-IgG antibody, followed by ECL Western blot detection reagents . Densitometer scans of blots were done using the AlphaImager 2000 documentation and analysis system . In some experiments, cells were pre-incubated with the selective p38 MAPK inhibitor SB203580 (10 µM), the MEK1/2/ERK1/2 inhibitor UO126 (10 µM), the Akt inhibitor VIII , the Raf1 inhibitor InSolution™ Raf1 Kinase Inhibitor I (1 µM), or the PLC inhibitor U73122 (3 µM) ; or the Ca2+ chelator BAPTA-AM™ (10 µM), the PI3K inhibitor wortmannin (100 nM), or the mTOR inhibitor rapamycin (50 µg/ml) . The concentration of the inhibitors used was determined to be optimal in preliminary studies that tested different concentrations of each inhibitor. The effect of the inhibitors on macrophage viability was assessed by trypan blue exclusion, and on bacterial viability by microbiologic analysis as previously described [Macrophages from C57BL/6 WT mice were pre-incubated with medium only, rapamycin (3 h) or with selective inhibitors (1 h) followed by stimulation with escribed ,44. Protescribed . The conF. tularensis LVS. Cells were then washed, lysed and assessed by Western analysis.Murine macrophage-like RAW 264.7 cells were cultured in 96- or 24-well plates in complete medium until 50-70% confluency was reached. Cells cultured in 96-well plates were transfected with nonspecific siRNA or with siRNA (100 nM) targeted to the Akt1/2 or PLCγ1 genes  using Lipofectamine™ RNAiMAX (0.3 µl), whereas cells cultured in 24-well plates were transfected with non-specific siRNA or siRNA targeted to the mTOR gene  using Lipofectamine™ RNAiMax (1.5 µl) transfection reagent , according to the manufacturer’s instructions. Transfected cells were incubated at 37°C for 5 days and rested for 4 h prior to the addition of F. tularensis LVS (MOI=20). Cells were washed twice with PBS and lysed with 500 µl of lysis buffer. Lysates were collected and assessed for protein content. Equal amounts of protein were transferred to microfuge tubes and 5 µl of anti-rictor antibody  or 5 µl of IgG control antibody  were added to the correspondent samples. Samples were rotated overnight at 4°C. Afterwards, 50 µl of protein A sepharose CL-4B , prepared in a 50% slurry, was added to each sample and incubated for 2 h. Samples were then washed 3 times in lysis buffer and 45 µl of Laemmli buffer with 5% mercaptoethanol was added to each sample and assessed by Western analysis.Macrophages from C57BL/6 WT mice were cultured in 60-mm cell culture plates overnight. Cells were pretreated with rapamycin  or media only, followed by infection with F. tularensis LVS (MOI=20). Cells were washed twice with PBS and lysed with 750 µl of TritonX buffer. Lysates were collected and assessed for protein content. Equal amounts of protein were transferred to microfuge tubes and 30 µl of 7-Methyl GTP sepharose  was added to each sample. The samples were rotated for 2 h at 4°C. Samples were then washed 3 times in TritonX buffer, and 45 µl of Laemmli buffer with 5% mercaptoethanol was added to each sample and assessed by Western analysis.Macrophages from C57BL/6 WT mice were cultured in 60-mm cell culture plates overnight. Cells were pretreated with rapamycin  or media only, followed by infection with 5 cells/well overnight in 24-well plates. Cells derived from WT mice were incubated or not with rapamycin , U73122 , UO126 , SB203580 , Akt VIII , wortmannin , BAPTA  or cytochalasin D  prior to incubation with bacteria. The indicated concentration of each inhibitor was shown to be optimal in preliminary studies. RAW cells transfected with control siRNA or with siRNA directed to the mTOR gene were cultured in complete medium in 24-well plates (see above). Freshly harvested F. tularensis LVS (MOI=20) were added to WT derived cells and to a portion of the transfected RAW cells for 90 min to assess bacterial invasion. Following incubation, cells were washed (5x) with PBS+ at room temperature and complete media containing gentamycin (50 µg/ml) was then added to the cultures for no more than 45 min to kill extracellular bacteria. Afterwards, cells were washed as described above and lysed for 5 min with ice-cold distilled water (150 µl). Lysates were serially diluted, plated on Mueller-Hinton II agar plates, and plates were incubated at 37°C in a 5% CO2 atmosphere [Thioglycollate-induced peritoneal macrophages from WT mice were harvested, washed and cultured in complete medium at 5 x 10mosphere . AdditioF. tularensis (MOI= 20), and then all sets were incubated for 90 min. Cells were then quickly washed with ice-cold phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde in PBS for 10 min at room temperature followed by permeabilization with 0.1% Triton X-100 in PBS for 7 min at room temperature. The coverslips were then incubated for 45 min at room temperature with Phalloidin conjugated to Alexa-Fluor 594  to stain actin and mouse monoclonal F. tularensis anti-lipopolysaccharide (LPS) antibody  to visualize F. tularensis bacteria. Cells were washed with 0.2% Tween 20 in PBS and incubated for 30 min at room temperature with goat anti-mouse Alexa-Flour 488 secondary antibody . Cells were washed with 0.2% Tween 20 in PBS, nuclei were labeled with Hoescht stain and coverslips were mounted in 9:1 glycerol/PBS with 0.1% p-phenylenediamine  on glass slides. Z-stack confocal images were acquired using a Nikon Eclipse TE 2000-U and Velocity 3D Image Analysis software .Cells were plated in 6-well plates with coverslips in each well and either left untreated or treated with rapamycin or wortmannin, as described above. One set of wells  was supplemented with buffer alone, while the other received t test using the InStat program .Statistical significance between infected control cells and infected cells pretreated with inhibitors was evaluated by Student’s two-tailed F. tularensis LVS, freshly isolated peritoneal macrophages were pretreated or not with the mTOR inhibitor rapamycin or with media containing DMSO (control). Cells were then co-cultured for 90 min with freshly harvested F. tularensis LVS and invasion was assessed. Rapamycin treatment of macrophages significantly reduced F. tularensis invasion to 19% compared to F. tularensis infected, untreated control cells  and that grown in Mueller-Hinton broth (MHB) [F. tularensis grown in BHI compared to those grown in MHB resulted in a similar phosphorylation pattern of mTOR downstream effector molecules, and a similar ability to invade macrophages. Therefore, macrophages were incubated with freshly harvested bacteria grown in BHI [F. tularensis grown in BHI or that grown in MHB  ,47. Sincn in BHI  or in MHn in BHI ,42 for 9n in MHB . F. tularensis infection, peritoneal macrophages were pre-treated or not with rapamycin and then exposed to Francisella for various times. The phosphorylation of mTORC1 downstream effector proteins was then assessed as previously described [Thr389 induced by F. tularensis LVS infection was seen at 30 min, and was increased at 60 and 120 min, whereas p70S6KThr421/Ser424 phosphorylation was detected at 10 min and peaked at 30 min  detected was notably less than that observed in non-rapamycin treated, infected macrophages  in the number of invading bacteria, compared to that seen with siRNA control transfected RAW cells  was achieved by use of the Akt inhibitor VIII in macrophages exposed to Francisella; however, essentially no change was seen in p70S6K phosphorylation at Thr389 or at Thr421/Ser424 or in the phosphorylation of 4E-BP1, eI-F4E or S6 at Ser235/236 or at Ser240/244 ethane-N,N,N’N’-tetraacetic acid, sodium (BAPTA) , and then lysed and subjected to Western analysis. Treatment of cells with U73122 or BAPTA prior to their incubation with F. tularensis LVS decreased the level of phosphorylated p70S6K (pp70S6K) at Thr389 and at Thr421/Ser424, as well as that of 4E-BP1 (p4E-BP1)  . Downregof BAPTA , whereasl 4E-BP1 . The rell 4E-BP1 . 2+ signaling pathways [E. coli [Francisella, resulted in downregulation of pp70S6K and p4E-BP1  or p38 (SB203580) signaling, exposed to bacteria, and then the number of invading Francisella assessed. Inhibition of MEK/ERK significantly decreased F. tularensis LVS host cell invasion to 63% compared to infected control cells .TLRs are pattern recognition receptors that can distinguish molecules broadly shared among microbial pathogens. Recognition of these molecules via TLRs triggers the activation of downstream signaling cascades. Since it had been previously shown that  agonist ,65, we nKO cells . AssessmKO cells . Interesrophages . Our finWT cells . These fegulated , suggestF. tularensis did not induce phosphorylation of p38 and ERK1/2 MAPK, whereas phosphorylation was seen in WT and TLR4KO macrophages at 10, 30 and 60 min following bacterial stimulation . Furthermore, inhibition of mTOR or of PI3K signaling by rapamycin or wortmannin, respectively, resulted in an alteration of the actin cytoskeleton architecture and inhibited the phosphorylation of mTOR downstream effector molecules. Moreover, in the presence of rapamycin or wortmannin F. tularensis invasion of primary macrophages was significantly reduced. Studies have shown that mTOR and downstream effector molecules are involved in actin cytoskeleton reorganization. For instance, inhibition of mTOR signaling using rapamycin or stable inhibition of raptor (mTORC1) and rictor (mTORC2), decreased actin cytoskeleton remodeling [C. sakazakii invasion of brain endothelial cells and prevented actin rearrangement necessary for invasion [Remodeling of the actin cytoskeleton is central to the invasion of host cells by bacteria, including that of modeling . Moreovemodeling  demonstrmodeling ,67. Furtmodeling , and botmodeling . The mTOmodeling ,53, a fiinvasion .F. tularensis in primary macrophages, and not surprisingly, siRNA to Akt or chemical inhibition of Akt did not abrogate phosphorylation of mTOR downstream effector molecules, including that of p70S6K. These observations suggested an Akt-independent mechanism for the phosphorylation of mTOR downstream signaling molecules. These findings gain support from studies demonstrating that phosphorylation of mTOR downstream effectors was Akt-independent in leukemic cells [While inhibition of rictor (mTORC2) decreased actin cytoskeleton remodeling , and Aktic cells , transfoic cells . HoweverFrancisella [F. tularensis LVS, revealed an apparent downregulation of ERK1/2 phosphorylation. This MAPK is the downstream effector of Syk and was implicated by Parsa et al. [F. tularensis host cell infection. Indeed, the MEK/ERK pathway has been implicated in host cell entry by other pathogens like C. pneumoniae [F. tularensis into host cells shown in the present study and that of others [Francisella invasion of peritoneal macrophages. The pathogen Rickettsia rickettsii has been shown to use p38 signaling for host cell entry [F. tularensis LVS infection of macrophages induced the phosphorylation of p38, but in the presence of rapamycin, as shown in this study and by others [F. tularensis invading macrophages, it also resulted in the downregulation of pp70S6K and pS6, and that the simultaneous inhibition of these signaling molecules, had an additive effect. The involvement of the MEK/ERK and p38 pathways in the regulation of the mTOR downstream effector molecules was not unexpected, since various studies favor a role for MAPK in the phosphorylation of the S/T-P (proline-directed serine/threonine) sites in the autoinhibitory C-terminal domain for the modulation of p70S6K [421/Ser424 in the C-terminus, consequently loosening the conformation and permitting the phosphorylation of Thr389 [Analysis of mRNA transcripts in rapamycin-treated cells demonstrated downregulation of several transcripts linked to phagocytosis . It is kncisella . Moreovea et al.  and suppeumoniae . In addif others , our finll entry . Furtherll entry . During ll entry . Along ty others ,68, altef p70S6K ,64. Actif Thr389 . Thr389 was abrogated by ERK1/2 and p38 inhibitors, while that at Thr421/Ser424 was blocked by ERK1/2, but not p38 inhibition [421/Ser424, but not at Thr389 was observed, and only the dual use of the inhibitors UO126 and SB203580 suppressed phosphorylation at these sites [235/236 seen in our studies is supported by the findings of Roux et al. [Ser473 phosphorylation, yet inhibition of the MEK/ERK and p38 pathways in cells exposed to Francisella resulted in an increase in the level of pAktSer473, as compared with that seen by the inhibition of p38 only. While it has been previously reported that p38 regulates AktSer473 phosphorylation via MK2 (MAPKAPK-2), and that inhibition of p38 by SB203580 downregulates this process [Ser473 upon suppression of the MEK/ERK and p38 pathways? Menges et al. [Ser473 (data not shown), hence supporting this notion. Given the reported involvement of p38 in the induction of G1/S and G2/M cell cycle checkpoints in response to stimuli [Ser473 phosphorylation, as observed in our studies.Differential requirements for ERK1/2 and p38 on p70S6K phosphorylation have been observed contingent on the stimuli. For instance, UV-irradiation induced phosphorylation of p70S6Khibition . In addise sites . These rx et al. , althougx et al. . Interes process ,78, how s et al.  showed t stimuli , inhibitE. coli [Francisella host cell entry when cells were pre-treated with the PLC inhibitor U73122. Since the cell permeable molecule BAPTA is enzymatically converted to the non-permeable, calcium chelator BAPTA inside the cell, only intracellular calcium is chelated [F. tularensis. Furthermore, this inhibition was associated with a downregulated phosphorylation of mTORC1 downstream effectors as shown in other systems [The involvement of phospholipases in phagocytic signaling has been well documented . PhosphoE. coli . The invchelated . Thus, o systems .E. coli by epithelial cells, and no bacteria could be recovered after pre-treatment with cytochalasin D or with antibodies against TLR4, suggesting that TLR4 participates in downstream signaling events that mediate bacterial internalization [Aspergillus fumigatus conidia, since infection of macrophages deficient in TLR2 rendered reduced conidial cell entry [F. tularensis LVS into primary macrophages, and, in the context of our investigations, the involvement of downstream effector molecules of mTOR, as well as signaling molecules associated with their regulation via TLR2 signaling, are important for this process. TLRs are highly conserved pattern recognition receptors and their recognition of pathogen associated molecular patterns induces the activation of host signal transduction pathways that result in an immune response that ultimately will aid in bacterial clearance . Howeverlization . Furtherll entry . ResultsF. tularensis LVS into primary murine macrophages through TLR2 signaling. Moreover, the phosphorylation of mTOR downstream proteins via PLC/Ca2+ and MAPKs signaling was also shown to play a role in Francisella invasion of macrophages  Peritoneal macrophages derived from WT mice were exposed to freshly harvested F. tularensis LVS (MOI=20) grown in Brain Hear Infusion broth (BHI) or in Muller-Hinton broth (MHB) for 90 min to assess bacterial invasion. (B) Peritoneal macrophages were exposed to F. tularensis LVS grown in BHI or MHB for 0-120 min and then lysed. Total p70S6K and phosphorylated p70S6K (Thr389 and Thr421/Ser424), 4E-BP1 (Ser65), S6 (Ser235/236 and Ser240/244) and eI-F4E (Ser209) were assessed by Western analysis. Samples analyzed contained an equal amount of protein. Unstimulated cells (time 0) served as negative controls. The gel is representative of three to five independent experiments.(TIF)Click here for additional data file.Figure S2Francisella and downregulates phosphorylation of mTOR downstream signaling cascade.mTOR siRNA decreased internalization of  (A) RAW cells were transfected with control siRNA or with siRNA to mTOR (100 nM), and after 5 days, cells were washed, rested and infected with F. tularensis LVS for 90 min to assess bacterial invasion. Values are the mean ± SEM of 5 independent experiments, each done in triplicate; **p < 0.001; *p < 0.05 compared with infected control transfected with control siRNA. (B) RAW cells were transfected with control siRNA or with siRNA to mTOR (100 nM) or non-transfected, and after 5 days, cells were washed, rested, infected with F. tularensis LVS (MOI=20) for 0-90 min and then lysed. Total p70S6K and Akt, and phosphorylated p70S6K (Thr389 and Thr421/Ser424), 4E-BP1 (Ser65), S6 (Ser235/236 and Ser240/244), eI-F4E (Ser209) and Akt (Ser473) were assessed by Western analysis. Samples contained equal amount of protein. RAW cells transfected with control siRNA were used as negative controls. Gels are representative of three to five independent experiments. (TIF)Click here for additional data file.Figure S3Phosphorylation of p70S6K in RAW cells transfected with siRNA to Akt1/2. RAW cells were transfected or not with siRNA to Akt1/2 (100 nM), and after 5 days, cells were washed, rested, infected with F. tularensis LVS (MOI=20) for 0 and 120 min and lysed. Total and phosphorylated p70S6K (Thr389) and Akt (Ser473) was assessed by Western analysis. Samples analyzed contained equal amount of protein. Unstimulated cells served as negative controls. Gels are representative of three to five independent experiments.(TIF)Click here for additional data file.Figure S4F. tularensis invasion of primary macrophages via TLR2 signaling.Proposed model of the signaling pathways involved in the phosphorylation of mTOR downstream effector molecules associated with (TIF)Click here for additional data file.


Yet a severe global glaciation during marine isotope stage (MIS) M2 interrupted this phase of global warmth ∼3.30 million years ago, and is seen as a premature attempt of the climate system to establish an ice-age world. Here we propose a conceptual model for the glaciation and deglaciation of MIS M2 based on geochemical and palynological records from five marine sediment cores along a Caribbean to eastern North Atlantic transect. Our records show that increased Pacific-to-Atlantic flow via the Central American Seaway weakened the North Atlantic Current and attendant northward heat transport prior to MIS M2. The consequent cooling of the northern high latitude oceans permitted expansion of the continental ice sheets during MIS M2, despite near-modern atmospheric CO2 concentrations. Sea level drop during this glaciation halted the inflow of Pacific water to the Atlantic via the Central American Seaway, allowing the build-up of a Caribbean Warm Pool. Once this warm pool was large enough, the Gulf Stream–North Atlantic Current system was reinvigorated, leading to significant northward heat transport that terminated the glaciation. Before and after MIS M2, heat transport via the North Atlantic Current was crucial in maintaining warm climates comparable to those predicted for the end of this century.The early Late Pliocene (3.6 to ∼3.0 million years ago) is the last extended interval in Earth's history when atmospheric CO The ∼3.3–3.0 Ma time slab known as the mid-Piacenzian Warm Period mPWP,  has beenInterrupting an interval of global warmth, MIS M2 has been proposed as an early, failed attempt by the Earth's climate to establish a pattern of intense and frequent Northern Hemisphere glaciations We established high-resolution palynological and geochemical records from five ocean drilling sites along a southwest–northeast transect in the North Atlantic covering the Caribbean Warm Pool, Gulf Stream, subtropical gyre and North Atlantic Current (NAC) over the interval 3.400–3.180 Ma to determine the role of ocean circulation in causing the extensive glaciation of MIS M2 . Our surhttp://doi.pangaea.de/10.1594/PANGAEA.804677. Previously published Mg/Ca and dinoflagellate cyst data http://doi.pangaea.de/10.1594/PANGAEA.758710 and http://doi.pangaea.de/10.1594/PANGAEA.758711.Samples were collected at the IODP Bremen Core Repository (Germany) and Gulf Coast Repository  from five sites constituting a transect between the Caribbean Sea (ODP Site 999), western North Atlantic (DSDP Site 603), and the eastern North Atlantic . The foraminiferal geochemistry data and palynomorph assemblages were acquired from the same samples at each of the five ocean drilling sites, and samples for biomarker  analysis were taken from the same sample depths at three sites. All generated dinoflagellate cyst and geochemical proxy data are accessible through the database PANGAEA at Lycopodium clavatum tablets were added before applying standard palynological preparation techniques involving cold HCl and HF acids Lycopodium clavatum counts and the dry weight of the <125 µm fraction Our laboratory technique allows dinoflagellate cysts and foraminifera to be extracted from the same samples  (herein O. centrocarpum) all reflect the presence of the North Atlantic Current (NAC). At DSDP Site 610 and IODP Site U1308, O. centrocarpum concentrations are highest also when the relative abundances are high, independently corroborating the value of O. centrocarpum as a NAC indicator species.The assemblage composition of dinoflagellate cysts in core-top samples is largely related to the present-day overlying water masses Full authorial citations of species discussed in the text are given in Globigerina bulloides (250–315 µm) for Sites 603, 610, U1308 and U1313 and five specimens per sample of Globigerinoides sacculifer (250–355 µm) for Site 999. Benthic foraminiferal isotope data are based on at least one >250 µm specimen of Cibicidoides wuellerstorfi or Uvigerina perigrina per sample. Cibicidoides wuellerstorfi δ18O values have been corrected by adding 0.64 ‰ Foraminifera were picked from the >125 µm dry fraction of each sample. Planktonic foraminifera isotope data were measured using a Finnigan MAT 251 mass spectrometer at the Isotope Laboratory, Geosciences Department, University of Bremen using five specimens per sample of G. bulloides (250–315 µm) or G. sacculifer (250–355 µm) and 20 specimens per sample of Globorotalia inflata (250–400 µm) from Sites 610, U1308 and U1313. The cleaning procedure for Mg/Ca measurements is described elsewhere 3, the samples were centrifuged and diluted for analysis on an ICP-OES (Perkin Elmer Optima 3300R) at the Geosciences Department, University of Bremen. The analytical precision of the Mg/Ca analyses for G. bulloides, G. sacculifer, and G. inflata combined was 0.17% (n = 459). Reproducibility based on replicate samples (n = 32) of both G. bulloides and G. sacculifer was ±0.11 mmol/mol (∼3.3%). The validity of analyses was checked by analysing an artificial in-house standard to monitor drift of the ICP-OES (Mg/Ca  = 2.93 mmol/mol) and the limestone standard ECRM752-1 (Mg/Ca  = 3.75 mmol/mol) to allow inter-laboratory comparison G. bulloides into SSTMg/Ca: Mg/Ca  = 0.52 exp 0.10 T Mg/Ca value of G. bulloides as spring to summer SSTs of the upper 60 m of the water column G. bulloides reflects the northward-migrating phytoplankton spring bloom in the North Atlantic Mg/Ca value of G. sacculifer represents the annual mixed-layer temperature of the upper 75 m of the water column for Caribbean Site 999 G. inflata calcifies throughout the water column, the SWTMg/Ca based on mostly non-encrusted G. inflata represents the temperature of the permanent thermocline G. inflata the error is estimated to be ±2–2.5°C For Mg/Ca measurements, we used 20–25 specimens per sample of 18Osw) was calculated via a standard formula 18O were measured on the same planktonic foraminiferal species, the possible effects of seasonality and habitat differences are minimised. We used the LR04 global benthic foraminiferal δ18O stack 18Osw, resulting in a δ18Osw-ice record that approximates local variations in salinity.The oxygen isotope composition of seawater  We generated high-resolution carbonate sand fraction data from ODP Hole 999A over the study interval. In addition, we used the available low resolution, long-term carbonate sand fraction record of the same site 3) were taken, avoiding visible mineral concretions and areas influenced by the coring process, from the working halves of Sections 603C-17X1 to 603C-18×1 (12 samples between 136.51 and 146.37 mbsf), Sections 610A-17H3 to 610A-17H4 (17 samples between 156.74 and 158.75 mbsf), Sections 610A-17H6 to 610A-18H1 (19 samples between 161.20 and 163.85 mbsf), and Sections U1308C-26H2 to U1308C-26H5 (20 samples between 230.48 and 239.45 mbsf). The discrete samples were measured at the Geosciences Department, University of Bremen on a cryogenic magnetometer (model 2G Enterprises 755 HR). The natural remanent magnetization (NRM) was demagnetized in nine steps (10–100 mT), and inclination and relative declination, and their confidence intervals were determined from line fits of straight-line segments in a Zijderveld diagram. Note that absolute declination depends on section and core orientations, whereas inclination values rely on the fact that the drill hole is to a very good approximation perpendicular. All ages of the magnetic reversals are according to the ATNTS 2004 The positions of magnetic reversals for the Mammoth Subchron in DSDP Holes 603C and 610A In DSDP Hole 610A, the upper boundary of the Mammoth Subchron was found between 158.35 mbsf (positive inclination) and 158.75 mbsf (negative inclination). The reversal at the base of the Mammoth Subchron is more difficult to identify due to a coring gap between Cores 610A-17H and 610A-18H, and disturbed sediment in the upper 25 cm of Section 610A-18H1 An age model was established for each hole –S4 by tu18O record to the astronomically dated benthic δ18O records from equatorial East Pacific ODP Site 846 18O stack The original age model of ODP Site 999 O. centrocarpum and such warm water dinoflagellate cyst taxa as Impagidinium aculeatum, I. paradoxum, I. patulum, I. solidum, and Polysphaeridium. zoharyi which are also present there today I. aculeatum, I. paradoxum, I. patulum, and Invertocysta spp. An active NAC brought warm waters (15.2–18.6°C in the uppermost 60 m) northward over Sites U1308 and 610  and the presence of  and 610 , expressSSTMg/Ca , 4, indi18Osw-ice) (Although deeper-water exchange via the CAS had been restricted since ∼4.6 Ma Osw-ice) , a low cOsw-ice) , and higOsw-ice) . The lowOsw-ice) , 5C highO. centrocarpum (our NAC tracer) remained present in low abundance. At the same time we find a peak abundance of O. centrocarpum , contemporaneous changes occurred in the North Atlantic surface circulation. At ∼3.315 Ma, a major reduction in northward flow of warm NAC waters is reflected at Sites 610 and U1308 by a major turnover of the dinoflagellate cyst assemblages within 1–2 kyrs, and an initial cooling of the surface waters is registered . This corocarpum  and surfrocarpum  at subtralkMg/Ca and largely unchanged dinoflagellate cyst assemblages at subtropical gyre Site U1313 is difficult to interpret . During the glacial conditions, subtropical gyre circulation persisted as attested by a continuance of the Gulf Stream at Site 603 where no major changes in the dinoflagellate cyst assemblages were recorded . The connterpret . During Bitectatodinium tepikiense, Filisphaera filifera, I. pallidum, Nematosphaeropsis labyrinthus, Pentapharsodinium dalei) at Site 610 and oligotrophic conditions  at Site U1308 (2) 18Obenthic values during MIS M2 compared to today  at Site 999 from the glacial maximum at ∼3.295 Ma onwards show that Caribbean surface waters became warmer and more saline while remaining oligotrophic . We provide a conceptual model based on palynological and geochemical records in the North Atlantic and Caribbean for glacial expansion and consequent deglaciation during an otherwise globally warmer world .2, and ice volume records; A long-term global cooling trend  to a volume seemingly larger than present, sea level fell to more than 10 m and possibly as much as 65 m below present . This clThe transition from MIS M2 to the mPWP can be seen as the evolution of a world with comparable global temperatures to present and slightly larger ice sheets, to a world with global temperatures ∼3°C higher than today and glaciation strongly diminished and localised in the Northern Hemisphere. Although operating on a longer time scale, this climate transition can provide valuable insights into the present anthropogenically-forced climate transition towards a globally warmer planet, being comparable to projections for the end of this century. In view of this projected climate warming, our results from the Late Pliocene show that high-latitude North Atlantic surface circulation and SSTs are a crucial factor in the expansion and contraction of Northern Hemisphere ice sheets.Figure S1Age model for DSDP Hole 610A based on the correlation of oxygen isotope records from the studied intervals with the LR04 benthic oxygen isotope global stack.Left panel: core-sections, polarity subchrons, including uncertainty interval for the exact position of each reversal of the Mammoth Subchron, and benthic isotope record against depth (mbsf). Middle panel: correlation of the benthic record  to the LR04 global stack of benthic isotope records Right panel: sedimentation rate based on our age model. Inset gives the tie points used, and correlation values of the benthic record running mean and raw data with the LR04 global stack. Note: Hole 610A shows a coring gap between Cores 610A-17H and 610A-18H, and sediment disturbance in the upper 25 cm of Section 610A-18H1.(TIF)Click here for additional data file.Figure S2Age model for IODP Site U1308 based on the correlation of oxygen isotope records from the studied intervals with the LR04 benthic oxygen isotope global stack.Left, middle and right panel and inset as for (TIF)Click here for additional data file.Figure S3Age model for IODP Site U1313 based on the correlation of oxygen isotope records from the studied intervals with the LR04 benthic oxygen isotope global stack.Left, middle and right panel and inset as for (TIF)Click here for additional data file.Figure S4Age model for DSDP Site 603 based on the correlation of oxygen isotope records from the studied intervals and palaeomagnetic reversals with the LR04 benthic oxygen isotope global stack.Left, middle and right panel and inset as for (TIF)Click here for additional data file.Figure S518O global LR04 stack Shown on the left are the benthic δ18O record of IODP Site 1241  compared to the benthic δ. On the right, the LR04 global stack is compared to the old 18O curve of ODP Site 999. The latter is a fine-tuning of the (TIF)Click here for additional data file.


The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO2-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating  and inhibiting (e.g. eIF4G phosphorylation) molecular events.Photosynthetic CO In addition to nitrogen nutrition, metabolic commitments and transcriptional control, mRNA translation is believed to be of importance to regulate protein synthesis and protein content of plant tissues. Plants are phototrophic organisms and thus their translational activity is strongly influenced by light and photosynthesis. Pioneering studies with isotopic labeling showed that in mature leaves, gross protein synthesis , since alterations of its phosphorylation level have drastic effects on growth and polysome formation Arabidopsis leaves in different photosynthetic contexts: various CO2 conditions (ordinary [CO2] (380 ppm), high [CO2] (1000 ppm), low [CO2] (100 ppm)) in the light and ordinary CO2 in the dark. We took advantage of a gas-exchange system with liquid N2 spraying for instant sampling and nanoLC-MS/MS based phosphoproteomics to characterize phosphorylated peptides in rosette leaves. We report 11 new phosphorylation sites in eIF proteins and 14 new sites in ribosomal proteins (RPs), describe significant CO2-dependent protein phosphorylation patterns and show concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. Our results suggest a key role of eIF and RP phosphorylation in photosynthesis-driven regulation of mRNA translation in leaves.As an aid in clarifying the nature and the phosphorylation of molecular actors involved in photosynthesis-driven translational control, we have investigated the phosphorylation of ribosomal proteins and initiation factors in Arabidopsis rosettes were placed in a purpose-built chamber connected to a gas-exchange system, as described in the Material and Methods. CO2 and water vapour (H2O) were monitored so as to calculate photosynthesis and transpiration. After four hours at a photosynthetic steady-state at the desired CO2 mole fraction, leaf rosettes were sprayed with liquid nitrogen and sampled. Sampling in the dark was carried out on dark-adapted leaves, that is, after two hours in darkness following 4 hours of steady photosynthesis at ordinary CO2 (380 ppm). Protein composition and phosphorylation were analysed by nanoLC-MS/MS. 2 on photosynthesis demonstrating that the four types of samples analysed here  corresponded to strongly different photosynthetic contexts. This CO2 effect was independent from water deficit since the water vapour draw-down between evaporation sites and atmosphere remained constant (2 fixation (photorespiration activity), was indeed higher at low CO2 , and underwent a SCX (Strong Cation Exchange) chromatography. Collected fractions were enriched in phospho-peptides by IMAC  and then analysed by nanoLC-MS/MS. The quantity of proteins was determined by direct analysis (no SCX and labeling). (Phospho)peptides were identified with X!Tandem 2 and dark treatments. Therefore, the changes in phosphopeptide abundance described below are strictly related to changes in the phosphorylation ratio.For proteomic characterization, protein samples were digested by trypsin and then analysed as described in http://phosphat.mpimp-golm.mpg.de/) and from 229ESLAK and DRRpS229EpS231LAK, respectively, 290MCGTTEYMAPEIVR). Other new sites located in eIFs are listed in 173LTR) and eIF2Bδ in 10 different Ser residues (Phosphopeptides identified in RPs and eIFs are listed in residues .P<0.04) by conditions: light/dark and/or photosynthetic activity (CO2 mole fraction). Their phosphorylation patterns are shown in 2). Most sites in eIFs were significantly affected by light/dark conditions, except for eIF4B1 at Ser 480; by contrast, this site was significantly affected by CO2 mole fraction. Most sites were positively influenced by CO2 mole fraction (i.e. there was a positive correlation between phosphorylation and photosynthetic activity), except for eIF4G, eIF4A and eIF5A  conditions.The phosphorylation status of eIFs and RPs is known to be crucial in regulating protein interactions and activity for translation initiation Arabidopsis leaves in the light and in the dark has further shown that RPP1A/B/C, RPS6A/B, RPP0B, RPS2C and RPL29A are phosphorylated and RPP1A/B/C, RPS6A/B, and RPL29A respond to light/dark conditions: RPS6A/B appeared to be significantly more phosphorylated in the light while RPP1A/B/C and RPL29A appeared to be slightly but insignificantly more phosphorylated in the light Introduction), the stimulation of translational activity is thus likely to be associated with a pronounced phosphorylation of RPs, thereby promoting the formation of the initiation complex.In eukaryotic cellular systems examined so far , cytosolic RPs have been found to be phosphorylated, allowing a fine control of translation. RPS6 activity seems to be controlled by phosphorylation , eIF4  and eIF5  proteins are phosphorylated and many phosphorylation sites responded significantly to light/dark and photosynthesis conditions . ImportaArabidopsis using sequence alignment with wheat (Ser 53) ArabidopsisA. lyrata, Populus trichocarpa, Glycine max and Vitis vinifera). eIF4G appeared to be phosphorylated in the N-ter region as we found phosphorylation sites at Thr 177 and/or Ser 178. However, this region is not associated with a clear function in translation Most phosphorylation sites in RPs described here  are noveConsidering the whole data set obtained here, clear phosphorylation patterns were observed in key actors of translation initiation  likely reflecting stimulation of daytime translation (compared to the dark). There is a considerable literature showing that nitrogen metabolism and nitrate assimilation occur during daytime in leaves (reviewed in in folio. This is probably of prime importance in situ (in the field) since changes in CO2 mole fraction occur quite frequently due to, for example, stomatal closure or diurnal CO2 changes in the ecosystem atmosphere. Some uncertainty nevertheless remains as to the targets of such a translational regulation: since the use of different eIFs modifies mRNA affinity 4E), the nature of mRNA selected for increased translation during photosynthesis is probably finely adjusted. Future translatomics (and polysome) analyses or 15N-labeling followed by protein-specific isotopic analyses would be required to describe the full translational picture of photosynthetising leaves.Our understanding of translation initiation in plants is still rather incomplete and further work is needed to disentangle the specific role of individual eIFs and their associated phosphorylation. In that regard, our model in Arabidopsis (Col-0 ecotype) plantlets were transplanted to individual pots and grown in a controlled environment (growth chamber) under 8∶16 h light/dark (short days) at an irradiance of approximately 100 µmol m−2 s−1, 20/18°C day/night temperature, 65% humidity and nutrient solution  twice a week. Gas-exchange were carried out with a purpose-built cuvette adapted to three Arabidopsis rosettes connected to the sample channel of the Li-Cor 6400 xt . Water vapour in inlet air was fixed (dew point temperature 11.6°C) with a dew-point generator Li-610 . Air temperature in the chamber was maintained with a water-bath. Leaf rosettes were separated from the below-ground part and soil of the pot by a plexiglass wall (with specific holes for collars) so as to avoid alteration of gas-exchange by soil and root respiration. The upper wall of the leaf cuvette was made of a tight polyvinyl chloride film allowing instant sampling by liquid N2 spraying. Photosynthesis was allowed to stabilise under the desired CO2 mole fraction (at 250 µmol m−2 s−1 PAR) and after 4 hours, rosettes were instantly frozen and stored at –80°C for further analyses. Rosettes sampled in darkness were collected after 4 hours at 380 ppm CO2 and 2 hours dark-adaptation.After sowing on potting mix, Leaf fragments were finely ground with liquid nitrogen. Protein extraction was carried out by using the TCA/acetone method. Briefly, the powder was incubated in a precipitating solution  for 1h at –20°C. After centrifugation (19 000 g), the pellet was rinced three times in 0.07% β-mercaptoethanol in acetone and spin-dried. It was then suspended in a solubilization solution made of 6 M urea, 2 M thiourea, 2% CHAPS (w/v) and 30 mM Tris-HCl pH 7.8 (60 µL/mg) and cell debris were eliminated by centrifugation. Total protein content was determined using the 2-D Quant-kit . 2 mg of proteins were reduced by adding DTT  and then alkylated by adding iodoacetamide . The samples were diluted to 1 M urea by adding 50 mM ammonium bicarbonate. Protein digestion  was performed at an enzyme/substrate ratio of 1∶30 (w/w) by overnight incubation at 37°C, and stopped by adding 1% formic acid (v/v).2H2]formaldehyde (labeling). Each sample was loaded on a separate SepPak C18 cartridge column  and washed with 0.6% acetic acid (v/v). SepPak columns were flushed seven times with 1 mL of the respective labeling reductive reagent ). Samples were eluted with 500 µL of 0.6% acetic acid (v/v) and 80% acetonitrile (v/v). All labelled dimethylated peptides were homogenized to form a reference sample, before being mixed with the unlabeled dimethylated peptides in a 1∶1 abundance ratio.Tryptic peptides were spin-dried and re-suspended in 1 mL of 5% formic acid (v/v). Stable isotope dimethyl labeling was performed according to the on-column procedure described by Prior to SCX, the dimethyl-labeled peptides were spin-dried and resuspended in 500 µL of solvent A (30% acetonitrile (v/v), 5% formic acid (v/v), pH 2.5). SCX was performed at 200 µL/min using Zorbax BioSCX-Series II columns  and a Famos autosampler (LC Packings). After sample loading, the first 17 min were run isocratically at 100% solvent A, followed by an increasing pH gradient using solvent B (30% acetonitrile (v/v), 5% formic acid (v/v), 540 mM ammonium formate, pH 4.7). Twelve SCX fractions per sample were automatically collected using an on-line Probot system (LC Packings).4OH, 30% acetonitrile) by centrifugation at 8200 g. Eluted phosphopeptides were dried and kept at –20°C until LC-MS/MS analysis.SCX fractions were dried and resuspended in 300 µL of solvent C ). Peptides were gently mixed with 80 µL of Phos-Select iron affinity gel (Sigma-Aldrich) and incubated for 2 hours using a tube rotator, as described by 18 PepMap 100, particles of 3 µm; column 75 µm i.d., 150 mm length; Dionex). Buffers were 0.1% formic acid in water (solvent E) and 0.1% formic acid in acetonitrile (solvent F). Peptide separation was achieved using a linear gradient from 5 to 30% F at 300 nL/min. Eluted peptides were analysed with a Q-Exactive mass spectrometer (Thermo Electron) using a nano-electrospray interface . Peptide ions were analysed using Xcalibur 2.1 with the following data-dependent acquisition steps: (1) full MS scan on a 300 to 1400 range of mass-to-charge ratio (m/z) with a resolution of 70000) and (2) MS/MS . Step 2 was repeated for the 8 major ions detected in step 1.On-line liquid chromatography was performed on a NanoLC-Ultra system (Eksigent). A 4 µL sample was loaded at 7.5 µL/min on a pre-column cartridge  and desalted with 0.1% formic acid in water. After 3 min, the precolumn cartridge was connected to the separating PepMap C18 column (stationary phase Chttp://www.thegpm.org/TANDEM). Cys carboxyamidomethylation and light and heavy dimethylation of peptide N-termini and lysine residues were set as static modifications while Met oxidation and phosphorylation of tyrosine, serine or threonine residues were set as variable modifications. Precursor mass tolerance was 10 ppm and fragment mass tolerance was 0.02 Th. Identifications were performed using the TAIRrelease 8 database (http://www.uniprot.org/http://www.arabidopsis.org/). Identified proteins were filtered and grouped using the X!Tandem pipeline v3.2.0 (http://pappso.inra.fr/bioinfo/xtandempipeline/). Data filtering was achieved according to a peptide E value smaller than 0.01. The false discovery rate (FDR) was estimated to 0.92%.Database searches were performed using X!Tandem CYCLONE Click here for additional data file.Table S2Phosphopeptides identified using nanoLC-MS/MS.(DOCX)Click here for additional data file.


Dictyostelium is frequently confronted with environmental changes likely to compromise survival. Here, we investigated whether the recently described arrestin-related protein AdcA is part of the cell response to stresses.Cell behaviour is tightly determined by sensing and integration of extracellular changes through membrane detectors such as receptors and transporters and activation of downstream signalling cascades. Arrestin proteins act as scaffolds at the plasma membrane and along the endocytic pathway, where they regulate the activity and the fate of some of these detectors. Members of the arrestin clan are widely present from unicellular to metazoa, with roles in signal transduction and metabolism. As a soil amoeba, Our data provide evidence that AdcA responds to a variety of stresses including hyperosmolarity by a transient phosphorylation. Analysis in different mutant backgrounds revealed that AdcA phosphorylation involves pathways other than the DokA and cGMP-dependent osmostress pathways, respectively known to regulate PKA and STATc, key actors in the cellular response to conditions of hyperosmolarity. Interestingly, however, both AdcA and STATc are sensitive to changes in the F-actin polymerization status, suggesting a common primary sensor/trigger and linking the stress-sensitive kinase responsive for AdcA phosphorylation to the actin cytoskeleton. We also show that STATc-dependent transcriptional activity is involved for the timely dephosphorylation of AdcA in cells under stress.Under osmotic stress, AdcA undergoes a phosphorylation-dephosphorylation cycle involving a stress-sensitive kinase and the transcription regulator STATc. This transient post-transcriptional modification may allow a regulation of AdcA function possibly to optimize the cellular stress response. Cells are continuously subjected to environmental cues that determine their behaviour in terms of motility, adhesion, growth and differentiation. Their capacity to sense and respond to external stimuli largely relies on membrane proteins including receptors, adhesion molecules, channels and transporters that function as sensors and signal transducers. In the cytoplasm, adaptor proteins from the arrestin clan play key roles in the signal relay, acting downstream of transmembrane spanning proteins at the plasma membrane or/and along the endocytic pathway. A well-characterized system is the coupling of β-arrestins to ligand-activated G-protein coupled receptors (GPCRs) allowing their temporal desensitization, their trafficking to specific endocytic compartments and the activation of secondary signalling cascades -5. Over Dictyostelium harbours 6 arrestin-domain containing proteins (AdcA to F). Their expression is highly regulated during the developmental cycle but the role of the Adcs at the different stages of the developmental program is completely enigmatic. In contrast to canonical visual and β-arrestins, the Dictyostelium Adcs contain additional functional domains besides the arrestin core, including lipid binding- and protein-protein interaction domains [Dictyostelium. At this stage, cells are faced with a broad range of environmental changes, ranging from hypo- or hyper-osmotic conditions to nutrient deprivation and their survival in such conditions relies on multiple and complex strategies. Hyperosmotic challenge activates an intricate transcriptionally regulated osmostress response [The social amoeba  domains . The Adc domains . The Adcresponse .Our data presented here provide evidence that AdcA undergoes a massive phosphorylation in response to a variety of stresses, including hypertonicity, and that AdcA dephosphorylation is partly dependent on a STATc pathway and correlates with cell recovery, raising the question of a possible role of AdcA in stress response.GFP  indicating that the tagged and overexpressed proteins behave as the endogenous AdcA. Maximal amount of band 3 occurred past 30 min post stress application  affected the electrophoretic pattern of the AdcA protein. We therefore examined the impact of various cellular stresses especially osmotic stress to determine the signal(s) responsible for this modification. In our assays, exponentially growing cells were centrifuged, resuspended in fresh nutritive medium and left to recover in shaking suspension for at least 1 h 30 prior to stress application. This protocol avoided, thereafter, any possible stress due to centrifugation  and nutrient depletion. In these conditions, in KAx-3 cells, AdcA was detected as a major higher mobility form and occasionally a minor slower form  responsible for AdcA shift, protein samples were treated with calf intestine phosphatase (CIP) to test the possibility of phosphorylation. As shown in Figure 2O2) but only weakly to a 50 μM Cd2+ treatment, indicating that AdcA is sensitive to a large variety of cell stresses  of total HΦGFP in the bands 2 + 3 at 30 min post-shock versus 81 ± 8% (n = 3) for the full-length AdcAGFP protein. This difference could possibly be attributed to the higher level of expression of HΦGFP and a limiting level of kinase activity. Alternatively, if the subcellular localisation of HΦGFP were distinct from that of AdcA, this could also interfere with the phosphorylation efficiency. Indeed, while AdcA is massively associated with the endocytic apparatus, the HΦ construct is mostly found in the cytosol [R491AGFP, that is unable to bind PI(3)P, fails to localize to the endocytic compartments [GFP, AdcAR491AGFP phosphorylation is partial, despite an expression level comparable to that of AdcAGFP, supporting a correlation between the endocytic localization and the phosphorylation level of the protein rather than a phosphorylation level mirroring the expression level of the constructs P-binding FYVE domain in the C-terminal part and a histidine-rich triplicated region that extends the protein N-terminally. To localize the site(s) of phosphorylation, the effect of 200 mM sorbitol was measured on truncated forms of AdcA, AdcAΔHΦ cytosol . Anotherartments . SimilarGFP protein were so far unsuccessful.The HΦ domain of AdcA contains 31 serines/threonines (and 2 tyrosine residues), precluding a systematic point-mutation approach to test their relative contribution to the phosphorylation pattern. Attempts to map the phosphorylated site(s) by mass spectrometry on the immunoprecipitated HΦDictyostelium cells multiply actively as single cells (vegetative state) and nutrient depletion triggers a developmental program leading to a multicellular structure containing starvation-resistant spores. In Dictyostelium, the consequences of a hyperosmotic stress have been well characterized on cells starved for a 1–4 h period of time and several effectors of the osmoresponse have been identified [2+-based pathways, to activate the stress-activated transcription factor STATc [In nutritive conditions, entified -26. Hypeor STATc ,21,26,27922 phosphorylated in our conditions within minutes following application of the osmotic stress  operate to trigger AdcA stress-induced response. In hyperosmotically-stressed starving cells, a peak of cAMP is detected, due to the activation of the histidine kinase DokA and the subsequent transient inhibition of the cytosolic cAMP phosphodiesterase RegA [dokA null mutation maintained the sorbitol-inducible phosphorylation of AdcA  did not interfere with the full phosphorylation of AdcA in response to sorbitol  was still responsive to sorbitol treatment, excluding a role for this Ser/Thr kinase  responsible for AdcA phosphorylation, we tested AdcA response to sorbitol in strains disrupted in candidate kinases. As expected from the absence of AdcA response upon 8-Br-cAMP addition, a deletion mutant in the PKA catalytic subunit  . We first tested the impact of this disruption on STATc activation. As shown in Figure adcA did not affect STATc’s tyrosine phosphorylation/dephosphorylation when compared to KAx-3 or its translocation to the nucleus, indicating that AdcA is not an essential intermediate in the pathway controlling STATc response.Given that both AdcA and STATc responded to similar stresses , we exacassette . The deldstC deletion  affected the phosphorylation status of AdcA. In the two dstC- and Ax-2 strains, the phosphorylation peak was similarly attained around 30 min post sorbitol stimulation but then, in the dstC mutant, the protein remained phosphorylated for an extended period of time as evidenced by the massive amount of band 3 still present at 150 min (42 ± 9% in dstC-vs 14 ± 6% in Ax-2)  leads to a constitutively active STATc [mycPTP3 to 200 mM sorbitol led to a response of AdcA that resembles that observed in the dstC null strain with a persistence of AdcA phosphorylation, several hours after shock . Taken together, the data support a functional link between STATc activity and AdcA dephosphorylation. However, while STATc could very well control the expression of a component of the AdcA dephosphorylation pathway, an additional level of regulation possibly acting on the balance between the activity of the AdcA-specific kinase and that of its phosphatase must be invoked to account for the absence of effect of constitutively activated STATc.Conversely, the ) Figure B and C. ve STATc . As showk Figure B. Such rk Figure D, suggesWe next undertook a characterization of the cell behaviour at the time of AdcA dephosphorylation. When subjected to a stress of 200 mM sorbitol, the cell rounding and actin rearrangement induced within the first minutes after shock were transitory. As illustrated on Figure adcA disruption modified cell resistance to stress and recovery. To test a possible contribution of AdcA to the osmoresponse, parental KAx-3 and adcA null cells were first subjected to increasing concentrations of sorbitol (100 to 400 mM) and cell growth was followed on a 96 h period of time. Addition of sorbitol affected cellular growth in a dose-dependent manner but similarly in adcA null and control cells suggesting that adcA deletion had no significant consequences on survival in hyperosmotic conditions (not shown). Analysis of the adcA null cell response in greater detail within the first four hours following a 200 mM sorbitol treatment confirmed that the absence of AdcA has no impact on cell recovery as indicated by the macroscopic morphological changes (not shown) and restoration of endocytosis within the same time frame as for KAx-3 cells  cGMP- and Caor STATc ,26,32 anor STATc ,24. Whetor STATc . Using kis work, ,29), an R491A construct, were less efficiently phosphorylated than AdcA. Alternatively, the hypothesis that folding of the soluble domains of AdcA and of AdcAR491A are different enough from that of AdcA to decrease the affinity for the kinase would perfectly accommodate a soluble kinase. It is thus impossible at this stage to make an informed guess as to which kinase is responsible for AdcA’s phosphorylation. In mild conditions of stress (100–200 mM sorbitol), AdcA phosphorylation is transient and its dephosphorylation is partly dependent on STATc. Given the transcriptional activity of STATc, a simple hypothesis is that STATc regulates the expression of a mediator of AdcA dephosphorylation such as the phosphatase itself or a regulator of the enzyme. Na and colleagues established by means of DNA microarray profiling the transcriptional profile of vegetative cells challenged with high sorbitol, and, among the differentially regulated genes, those that were dependent on the presence of STATc [The identification of the kinase responsible for AdcA’s phosphorylation would clearly help positioning AdcA in the signaling cascade. In addition to GbpC, our data disqualified PKA, Erk1, Erk2 and MEK1 as key kinases in AdcA phosphorylation. The amoebal kinome includes 255 kinases, with 155 putative Ser/Thr-directed kinases . As AdcAof STATc . The lisadcA deletion on cell response to hyperosmolarity would rather argue against a role for AdcA as an actor of the osmostress response. However, to provide a definite conclusion on the significance of AdcA phosphorylation in the stress context, identification of the phospho-sites and the subsequent construction of phospho-mimicking and non-phosphorylable mutants of the protein will be essential. We showed that, in various contexts of stress affecting cell morphology and endocytic activity, AdcA response consistently correlates with cell behaviour. AdcA being a protagonist of the endocytic pathway, its phosphorylation could modify its function and interfere with the regulation and fate of some key membrane targets (e.g. ion transporters) involved in the stress response. While the absence of AdcA may be silent, its phosphorylated and/or dephosphorylated form could be necessary intermediates for an optimal response and reaching of a novel homeostasis  stressors described to trigger AdcA phosphorylation all alter the cellular morphology and (ii) cytochalasin A as well as latrunculins are able to induce AdcA response in the absence of any other stressor. Given the intricate network of interactions between cytoskeletal elements and transmembrane or membrane-associated proteins, remodelling of the actin cytoskeleton could directly contribute to the activation/inhibition of signaling cascades and the transmission of signals to downstream stress-response effectors, among which AdcA kinase.The responsiveness of AdcA to various types of stress raises the question of the nature of the cellular trigger common to all the stresses, and able to elicit AdcA phosphorylation. In eukaryotes, defects of the cytoskeleton are one of the major damages observed in response to stress conditions. In Dictyostelium, hypertonicity triggers immediate morphological changes with remodelling of the cytoskeleton and activation of a transcriptional response affecting the expression of hundreds genes. Our data identified a novel target of the osmostress response, the arrestin-related protein AdcA, the regulation of which by a phosphorylation/dephosphorylation cycle might allow an optimal response of cells under stress.In the telluric amoebae Dictyostelium discoideum parental strain KAx-3. The null cells gbpC- (DBS0302680), mek1- (DBS0236541), pkacat- (DBS0236783) and gca-/sgc- (DBS0236000) were obtained from the Dicty Stock Center. The erk1-and erk2- null cells were kindly provided by J. Hadwiger and the dokA- mutant was obtained from S. Schuster. The adcA-  and strains overexpressing AdcA constructs (G418R)  were described previously [dstC-  was described earlier [mycPTP3 and KAx-3/mycPTP3ΔC649S were obtained by electroporation in KAx-3 cells of the mycPTP3 and mycPTP3ΔC649S overexpressing constructs (G418R) derived from the work of the Firtel laboratory [Dictyostelium strains were grown in Petri dishes or in shaking culture at 21°C in the presence of appropriate antibiotics in maltose-containing axenic medium [dstC- that were grown in HL5 medium  supplemented with 14 g/l glucose.Except when mentioned, the experiments were conducted on the eviously . The dsttructs G4R phenylhydrazone , CdCl2 and H2O2 (Sigma-Aldrich) at concentrations indicated in the text. For treatment with sorbitol, glucose, NaCl and glycerol, 10× stocks diluted in appropriate medium depending on the experiment (nutritive medium or phosphate buffer) were used. For heat shock experiments, cells were transferred to a 32°C shaking water bath. Samples of 200 μl were removed just prior (t0) and during stress application as a function of time. Cells were rapidly centrifuged, resuspended in denaturing buffer  and boiled for 3 min at 94°C. To test the contribution of the cytoskeleton, cells were treated with 5 μM latrunculin B (Sigma-Aldrich) or 10 μM cytochalasin A (Sigma-Aldrich) and DMSO was used as control. 8-Br-cAMP (20 mM) , 8-Br-cGMP (20 mM) (Enzo) and thapsigargin (10 μM)  were used to test the impact of an increase in cAMP, cGMP and an elevation of intracellular calcium respectively. For experiments performed on adherent cells, stress was applied after 1 h 30 of recovery, by removal of the nutritive medium and addition of fresh medium containing the appropriate concentration of stressor.Cells were harvested in log-phase from shaking cultures and resuspended in nutritive medium at 105 cells were separated by SDS-PAGE and transferred on PVDF membranes. AdcA was detected using the guinea-pig anti-AdcA antibody (COB25) [Protein samples corresponding to 2 × 10 (COB25)  in TBS-0 (COB25) . The ant7 cells/ml in axenic medium. FITC-dextran  and sorbitol  or cytochalasin A (10 μM) were added simultaneously to the suspension. Samples of 107 cells were removed as a function of time and transferred in ice-cold axenic medium containing the same concentration of sorbitol. After 3 washes, cells were resuspended in 1 ml of 40 mM Mes-Na pH 6.5, counted rapidly and lysed by addition of 2 ml of 100 mM NaPi-0.25% (v/v) Triton X-100, pH 10. The fluorescence intensity was measured at λex 470 nm and λem 520 nm. Data were converted into an equivalent volume of internalized fluid using a calibration curve [Cells were resuspended at the concentration of 10on curve .Cells were let to adhere on glass coverslips for 1 h 30 in axenic medium and subjected to stress treatment as indicated. For live cell imaging, cells were observed directly by DIC or fluorescence microscopy. In that latter case, LOFLO nutritive medium (Formedium) was used because of a reduced autofluorescence compared to the original medium. For immunostaining, cells were fixed in axenic medium containing 4% paraformaldehyde (PFA) and 0.1% Triton X-100 for 10 min. After washes in PBS and 1 h incubation in PBS-0.5% BSA, cells were then incubated with the anti-STATc (7H3) antibodies in PBS-0.5% BSA at room temperature for 1 h, washed three times in PBS-0.1% Tween 20 and incubated with Alexa 488-coupled secondary antibodies  for an additional hour. Actin was stained using Alexa 594-phalloidin (Molecular Probes). Cells were observed directly after three washes in PBS-Tween. Images were acquired on an Axiovert 200 M Zeiss microscope equipped with a Piezo unit. The Axiovision software was used for image acquisition, deconvolution of z-stacks and maximal intensity z-projections.5 cells was diluted 20-fold in 50 mM Tris–HCl pH 8.8, 0.66 mM MgSO4, 1 mM MgCl2, 0.1 mM ZnCl2 and 1 mM spermidine. About 15 units of CIP were added and dephosphorylation was performed at 37°C for 30 min. For each time-point, an aliquot receiving no enzyme was treated in parallel as a control. Proteins were precipitated by a 1 h-incubation on ice after the addition of 10% trichloroacetic acid (TCA), centrifuged at 16 000 × g and resuspended in denaturing buffer containing 200 mM Tris base. Dephosphorylation was assessed by the abolition of the electrophoretic mobility shift on 8% SDS-PAGE gels.Dephosphorylation assays using the CIP phosphatase  were performed on SDS denatured samples. An aliquot corresponding to 2 × 10GPCR: G-protein coupled receptor; ART: Arrestin-related trafficking adaptor; Adc: Amoebal arrestin-domain containing protein; Arrdc: Mammalian arrestin-domain containing protein; CIP: Calf intestine phosphatase; PFA: Paraformaldehyde.The authors declare no conflict of interests.CH, GK and LA designed the study. CH, GK and LA conducted the experiments. CH, GK, TA, JGW and LA drafted the manuscript. All authors read and approved the manuscript.Movie 1. KAx-3 cells response to 200 mM sorbitol. KAx-3 cells were let to adhere on a glass coverslip in nutritive medium and subjected (t=-30 sec) to 200 mM sorbitol. The movie was taken by time-lapse video microscopy using DIC optics and a 63x objective. The time frame of the movie corresponds to approximately 9 min, with images taken every 10 seconds.Click here for file