Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr  Previous studies revealed vital roles for the Poly(ADP-ribose) Polymerase (PARP) protein in developmental program regulation, as well as in chromatin loosening and transcriptional activation. The main gap in our understanding of PARP protein roles during development involves integrating the biological activities of PARP protein into a general network of nuclear protein dynamics and gene expression. Here, we report the functional association of PARP protein activity with proteins trafficking through Cajal bodies. Cajal bodies (CBs) are nuclear organelles that regulate the biogenesis of RNA-protein complexes involved in transcription and splicing. We demonstrate that (1) PARP is present in CBs, (2) its loss leads to CB disassembly, (3) an increase in PARP levels leads to formation of CBs, and, finally, (4) PARP localization is ecdysone sensitive. We further propose a model in which PARP controls protein trafficking through the CB and contributes to CB formation. Specifically, our data show that chromosomal PARP1 molecules become activated by developmental cues, such as ecdysteroid signaling, and are automodified by pADPr. Automodified PARP1 then binds protein components of nucleoli and chromatin. Following that, automodified PARP1 serves as a “shuttle” for protein delivery to Cajal bodies for recycling, thus contributing to protein trafficking through CBs and to the overall stability of Cajal body.Drosophila development, we used Drosophila stock ubiquitously expressing PARP1-DsRed recombinant protein. Previously, we biologically validated this construct by testing its ability to rescue a CH1Parp mutation phenotype and by using immunofluorescence to assess recombinant protein localization to chromatin DrosophilaDrosophila tissues demonstrated strong colocalization of pADPr and Coilin protein  to precipitate Coilin-containing protein complexes. We prepared total protein extracts using Drosophila PARP1 mutant stock ubiquitously expressing PARP1-ECFP or PARPe-EGFP transgenic constructs. First, we found that PARP1-ECFP, as well as PARPe-EGFP, proteins always co-purify with Coilin, as well as Fibrillarin protein . A significant amount of Coilin protein was co-purified with PARP1 from wild-type osophila , while tPARP1 mutant cells PARP1 mutants. In wild-type animals, Coilin protein localizes exclusively in CB , and the HLB/CB markers are always colocalized with PARP1-DsRed protein (in situ hybridization (FISH) using a U85 scaRNA probe. Using the U85 scaRNA probe demonstrated perfect colocalization with PARP-positive particle in 27.1parg mutant tissues , indicating that ectopic PARP1-DsRed expression does not modify 27.1parg mutant phenotypes. We next tested whether other components of CB also localize into ectopic CBs in Parg mutants. We found that subunits of RNA Polymerase II accumulate in those bodies . We dissected salivary glands from third-instar larvae of wild-type and e nuclei . Howeverreatment . Under tleoplasm . These r27.1parg mutant phenotypes. To accomplish this, we expressed a previously constructed epitope-tagged version of UASt::PARG-EGFP transgenic construct 27.1parg mutant animals, along with PARP1-DsRed. When expressed using constitutive 69B Gal4-driver 27.1parg lethality. Using temperature-inducible hsp::Gal4 activator , we were also able to suppress 27.1parg phenotypes. When expression of hsp::Gal4 was induced before third-instar larvae stage, the PARG-EGFP protein was strongly accumulated in nucleoplasm  (Multiple nucleus) , arrow. nucleus) , Inset. nucleus) . Howevernucleus) . To confPARP1 mutant nuclei  are small nuclear organelles detected in the nuclei of many eukaryotes in vivo.In summary, we have demonstrated that poly(ADP-ribosyl)ation is required for protein delivery into CB. Furthermore, interaction of the key protein Coilin with other protein components of CB, such as Fibrillarin, depends upon the activity of PARP1 and PARG proteins. These results provide the first molecular explanation for the roles that PARP1/PARG protein machinery play in the regulation of nucleolar organelle biogenesis and dynamics. Finally, our findings suggest the novel role of CB in nuclear protein recycling and emphasize the importance of increasing our knowledge about the properties of poly(ADP-ribose) dynamics 27.1Parg was constructed by Hanai et al. Drosophila larvae were heat-shocked for 1 hr at 37°C twice daily for 2 days prior to the second-instar stage. Balancer chromosome carrying Kr::GFP, i.e., FM7i, P{w1, Kr-GFP} 27.1Parg.Genetic markers are described in FlyBase Drosophila total cDNA as a template for PCR. The resulting PCR products were cloned through The Drosophila Gateway™ Vector Cloning System (Carnegie Institution of Washington) into a corresponding vector for Drosophila transformation. To make a catalytically inactive PARG-EGFP transgenic construct, two evolutionarily conserved Drosophila PARG sequence) were mutated to alanines, generating UAS::PargAA-EGFP transgenic construct. Transformation was as described in To construct UAS::PARP1-ECFP, we generated full-length PARP1 ORF using PCR. Primers were used: Parp1d, CACCatgtctgattctgcagttg, and Parp1r, ctttttggcagccgtag. We used wild-type Lysates for immunoprecipitation were prepared as follows: 15 third-instar larvae were collected for each sample. They were put into Eppendorf tubes and rinsed 3 times with 1 ml of dist. water. 500 ul of ice-cold lysis buffer  and 0.1 mM Pefabloc SC (Fluka)) were added to each tube, and larvae were homogenized by hand pestle homogenizer on ice. After 30 min incubation on ice, samples were centrifuged at 14500 RPM for 20 min (4°C). Supernatants were transferred to new Eppendorf tubes on ice. For one immunoprecipitation reaction, 500 mkl of total lysates were incubated with 25 mkl Protein-G Sepharose 4B (Sigma #P3296-5ML) on a rotating platform for 1 hr 30 min at 4°C. Beads were removed by spinning 1 min, 2000 g. Appropriate amount of antibody was added to the lysates, and the mixture was incubated 4 hrs on a rotating platform at 4°C. The following antibodies were used for immunoprecipitation: anti-Coilin  4, dehydrated in ethanol and propylenoxide, and embedded in EMbed-812  in flat molds. After polymerization for 60 hours at 65°C, 70 nm sections were cut on Leica Ultracut E microtome , placed on collodion/carbon-coated grids, and stained with 2% uranyl acetate/ lead citrate. Sections were viewed on a Tecnai 12 transmission electron microscope (TEM) .For ultrastructural analysis, the salivary glands were dissected, fixed in 2% formaldehyde/ 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, post-fixed in 1% OsO2, 10 mM EGTA, pH 6.9), cryo-protected in 2.3 M sucrose, mounted on aluminium pins, and frozen in liquid nitrogen. Thin frozen sections were then cut on a Leica EM UC6/FC6 cryomicrotome , collected on a drop of sucrose/methylcellulose mixture and placed on a formvar-carbon grid. The sections were labeled with primary antibody, and the label was subsequently visualized by colloidal gold conjugated to Protein A. Sections were stained/embedded in 2% methylcellulose/0.2% uranyl acetate and observed under a Tecnai 12 TEM.For EM immunocytochemistry, samples were prepared according to Tokuyasu 1980 For immunofluorescence, frozen samples prepared for TEM were cut to 0.5 µm-thick sections and labeled with primary antibodies followed by secondary antibodies conjugated to fluorochromes. Sections were washed in 200 mkl drops of 1XPBS+0.1% glycin solution 5 times for 1 min (Coverslip was placed face down on a drop), then blocked in 1XPBS+1%BSA twice for 3 min and incubated with primary antibody dissolved in blocking solution in humid chamber for 1 h. Afterwards, sections were washed with 1XPBS (4 times for 2 min) and incubated with secondary antibody in a humid chamber for 40 min. The mount was then washed with 1XPBS (3 times for 5 sec and 4 times for 2 min) and mounted in Vectashield . Samples were examined by confocal microscopy using a Leica TCS-LT microscope. The primary antibodies used were as follows: mouse monoclonal (mAb) H1  (1∶400 or 1∶1000), and pAb PAR  (1∶5000). Mouse Alexa-488, rabbit Alexa-568 and rabbit Alexa-633 (Molecular Probes) were used as secondary antibodies (1∶400). For DNA staining, propidium iodide was used at 0.05 mg/ml final concentration in Vectashield or Draq5™ , and up to 2.5 µM was added to the final wash with 1XPBS for 2 min before mounting in Vectashield.in vitro transcription reaction from genomic PCR amplified product as described previously 5′ TAGGGCGGAATTCATTAACCCTCACTAAAGGGATGCCCATGATGAAATATTCGAC3′ TAGGGCTCTAGATAATACGACTCACTATAGGGATCCTTGCGCTCAGATTACTAAAGACG.Fluorescent U85 scaRNA probe labeled with Alexa-488-UTP was prepared by Since this RNA probe was generated using polymerase T3 transcription, T7 polymerase was used for antisense. Probes were diluted in hybridization solution . Hybridization was performed overnight at 42°C.Figure S1Drosophila nuclei. The size and localization of these particles suggest certain similarities to Cajal bodies (CBs) and the related organelle, histone locus body (HLB). A. The dissected salivary glands expressing PARP1-DsRed (red) transgenic construct were stained with the DNA binding dye Draq5 (green). Position of extrachromosomal particles is indicated with arrows. N - nucleolus. B. The dissected salivary glands from wild-type Drosophila were fixed and partially squashed on a slide, followed by immunostaining with anti-pADPr antibody (red) and with the DNA binding dye DAPI (blue). Position of extrachromosomal particles is indicated with arrowheads. N - nucleolus.Small PARP1- and pADPr-positive extrachromosomal particles detected by confocal microscopy in (3.62 MB TIF)Click here for additional data file.Figure S2Drosophila stock (negative control) and stock expressing PARPe-EGFP were used. To detect PARPe-EGFP protein on Western blots, mouse monoclonal anti-GFP antibody was used. B. Nuclear GFP protein does not interact with Coilin. Co-immunoprecipitation assays using rabbit anti-GFP antibody. Drosophila stock expressing Nuclear-GFP protein was used. To detect proteins on Western blots, the following antibodies were used: mouse anti-GFP; Guinea Pig anti-Coilin and mouse anti-Actin. C. Preimmune serums from rabbit (Rab IgG), Guinea Pig (GP IgG) and mouse (Mab IgG) do not cross react with components of Cajal and Histone locus bodies. Co-immunoprecipitation assays using preimmune serums. Wild-type (WT) Drosophila stock and stock expressing PARPe-EGFP were used. To detect proteins on Western blots, the following antibodies were used: mouse anti-GFP; Guinea Pig anti-Coilin, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C. D. Intrinsic PARP protein interacts with coilin. Immunoprecipitation assays using rabbit anti-PARP1 and anti-GFP antibodies. Wild-type Drosophila stock was used. To detect proteins on Western blots, the following antibodies were used: Guinea Pig anti-Coilin and mouse anti-Actin.Control experiments confirm the specificity of PARP protein interaction with proteins of Cajal body. A. Fibrillarin interacts with PARP protein in vivo. Co-immunoprecipitation assays using rabbit anti-Fibrillarin antibody. Wild-type (WT) (0.35 MB TIF)Click here for additional data file.Figure S3Drosophila development in wild-type (wt) and Parg27.1 mutant animals. Northern blot hybridization using DsRed (to detect transgenic construct expression), PARP1  and Tubulin (loading control) DNA probes.PARP1 gene and PARP1-DsRed transgene expression during (0.24 MB TIF)Click here for additional data file.Figure S4Parg27.1 mutant (B) salivary gland nucleus and diploid Parg27.1 mutant nucleus (C) are shown. The arrows indicate nucleoplasmic bodies which are specific for the Parg27.1 mutant.The inhibition of PARG function induces ectopic CBs. (A–C) The electron micrographs of wild-type (A) and (1.75 MB TIF)Click here for additional data file.Figure S5Parg27.1 mutant nuclei. Immuno-gold staining of ectopic CB particles using (A) antibodies against Pol II CTD domain and (B) anti-rpb4 subunit antibody. Specific localization of immuno-gold staining (black dots) in CB is clearly demonstrated.RNA Polymerase II subunits are localized into ectopic CB in (1.09 MB TIF)Click here for additional data file.Figure S6Parg27.1 mutant , third-instar larvae, expressing PARP1-DsRed (red), were cultured in the presence  or absence (C) of ecdysteroid hormone (20E), followed by confocal microscopy. DNA was visualized with Draq5 staining (green). Nucleoplasmic PARP-containing bodies induced in Parg27.1 mutant nuclei (B) are indicated with an arrow. N - nucleolus.Ecdysteroid hormone controls PARP1 protein localization in vitro. Salivary glands from wild-type (A) and (3.26 MB TIF)Click here for additional data file.Figure S7Parg27.1 mutant nucleus is shown. The “free” nucleoplasmic (arrow) and “chromatin-embedded” (arrowhead) particles are shown. Inset. The cross section through the single CB-like particle is magnified . (B–D) Immuno-gold staining of “free” nucleoplasmic particles using antibodies (1) against pADPr (B), (2) against DsRed (C) and (3) against H1 (D) is shown. (E–G) Immuno-gold staining of “chromatin-embedded” particles using antibodies (1) against pADPr (E), (2) against DsRed (F) and (3) against H1 (G) is shown. Arrowhead indicates CB itself; arrow indicates surrounding materials accumulating on pADPr (E), but not PARP1 (F).The inhibition of PARG function induces ectopic CBs and relocation of PARP1 protein from chromatin into CBs. (A) The electron micrograph of (3.98 MB TIF)Click here for additional data file.Figure S8Drosophila S2 cells. Co-immunoprecipitation assays using mouse antibody against pADPr (10H). S2 cell culture was used to prepare protein extracts. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C.Key proteins of Cajal body and Histone locus body interact with pADPr in (0.17 MB TIF)Click here for additional data file.


C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts.The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a  S. cerevisiae Ste20, activates MAP kinase signaling in response to mating pheromone as a MAP kinase kinase kinase kinase (MAP4K) upstream of the MAP3K Ste11p C. elegans, one Drosophila, and multiple vertebrate/mammalian proteins The Ste20-related germinal center kinases comprise a large protein family that has been implicated in cellular processes ranging from cytoskeletal dynamics and stress responses, to cell growth, proliferation, and death Like the founding kinase Ste20, members of the GCK I, II, III, IV, V, VII, and VIII subfamilies have been reported to activate MAP kinase signaling, including JNK, p38, and ERK pathways C. elegans, ERK is activated at two distinct times during oocyte development: first, as germ cells progress through pachytene, and second, in maturing diakinetic oocytes residing proximal to the spermatheca C. elegans ERK ortholog MPK-1 and other components of the ERK cascade results in sterility characterized by the failure of germ cells to progress through pachytene In addition to intracellular events, MAP kinase signaling pathways regulate a remarkable number of developmental processes C. elegans oocyte maturation is regulated by sperm It has been recognized for a number of years that C. elegans member of the GCK III subfamily. Loss of GCK-1 results in the hyper-activation of a specific MAP ERK kinase isoform, abnormal oocyte development, and increased germ cell death. Altogether, this study has uncovered a novel role for a germinal center kinase in germ cell development and reveals that GCK kinases can have opposing roles in the regulation of MAP kinase activation and apoptosis.Here we report that MAP kinase activation in pachytene is inhibited by GCK-1, the sole C. elegans genome encodes 8 recognizable members of the germinal center kinase family C. elegans development, dsRNA corresponding to the full-length gck-1a isoform was microinjected or fed to wild-type (wt) young adult or L4 larval stage hermaphrodites. gck-1(RNAi) by either method resulted in progressive sterility. The animals were completely sterile within 24 hours of RNAi induction. gck-1(RNAi) hermaphrodites produced broods of <70 progeny as compared to >250 progeny from untreated wt age-matched controls. The F1 progeny of gck-1(RNAi) animals were completely sterile, and displayed a phenotype that was indistinguishable from their sterile mothers (below and data not shown). Consistent with these results, hermaphrodites homozygous for a gck-1 deletion allele that lacks a significant portion of the kinase domain (gck-1(km15)) (gck-1(lf), except as noted.The 1(km15))  had a siC. elegans hermaphrodite gonad consists of two U-shaped arms that share a common uterus (The n uterus . Beginnigck-1(lf) animals, gonads dissected from wt and gck-1(RNAi) hermaphrodites were examined by differential interference contrast (DIC) microscopy (gck-1(lf) animals had little to no discernable rachis, and the proximal ends were filled with dozens of small germ cells  germline defects, gonads dissected from wt, gck-1(RNAi), gck-1(km15) heterozygotes, and gck-1(km15) homozygous animals were fixed, stained with DAPI, and examined by deconvolution microscopy (gck-1(km15)/nT1, gck-1(RNAi), and gck-1(km15) gonads had progressively fewer germ cells than wt (gck-1(km15)/nT1 heterozygotes, gck-1(RNAi), and gck-1(km15) gonads had 35, 60, and 81% fewer pachytene nuclei respectively than wt  and gck-1(km15) gonads (gck-1(lf) gonads also contained a large number of nuclei whose stage in meiotic prophase could not be readily determined by morphology or position (To characterize croscopy . This an than wt . Differe) gonads . gck-1(lposition .gck-1(km15)/nT1 heterozygotes contained fewer germ cells, all other aspects of oogenesis were grossly normal and these animals were fertile (gck-1(RNAi) and gck-1(km15) animals, striking morphological differences from wt became apparent during pachytene (gck-1(lf) nuclei with paired, thread-like chromosomes were not arranged in an orderly pattern on the gonad periphery, and there was no clear transition from pachytene into diplotene  (gck-1(km15)/nT1 heterozygotes (gck-1(lf) hermaphrodites contained a large number of small cells/oocytes (gck-1(lf) gonads (Although the pachytene region of  fertile . Howeverachytene . Unlike ad arms) . Moreoveozygotes , arrows,/oocytes . While s/oocytes , arrows,) gonads .rrf-1(pk1417) hermaphrodites were fed control and gck-1 dsRNAs. RRF-1 encodes an RNA directed RNA polymerase that is required for RNAi in somatic cells but not the germline rrf-1(pk1417) mutant animals, but is still potent in the germline. DAPI staining revealed that the gck-1(RNAi) phenotype does not differ between wt and rrf-1(pk1417) gonads  gonads , indicatgck-1(lf) gonads display other characteristics of diakinetic oocytes, wt and gck-1(RNAi) gonads were immunostained for two markers that delineate diakinetic oocytes from earlier stages of meiotic prophase. Staining of wt hermaphrodite gonads with antibodies that recognize a phosphorylated residue on the C-terminal domain (CTD) of RNA polymerase II (H14), and histone H3 phosphorylated at serine 10 (pH 3) results in a specific pattern during C. elegans oogenesis To ascertain if the abnormal diakinetic-like oocytes in gck-1(lf) gonads was associated with all germ cell nuclei from the distal mitotic region through pachytene. However, in the proximal gonad, some of the cells displayed chromosomal H14 staining while in others, it was clearly cytoplasmic (gck-1(lf) germline.As in wt animals, H14 immunostaining in gck-1(lf) mitotic germ cell nuclei (gck-1(lf) proximal gonad, pH 3 immunostaining was not limited to oocytes residing next to the spermatheca, but was associated with the diakinetic-like chromosomes of cells scattered throughout the proximal half of gck-1(lf) gonads  gonads .C. elegans germline gck-1(lf) gonads.A canonical MAP kinase pathway regulates proliferation, development, and apoptosis in the gck-1(km15)/nT1 gonads with an antibody specific for activated MAP kinase (MAPK-YT) resulted in the expected pattern of MAP kinase activation during pachytene (gck-1(km15) animals there was a statistically significant increase in activated MAP kinase in pachytene compared to wt (gck-1(km15) proximal gonad  and differ only by the presence of an additional 5′ exon in the larger isoform Two isoforms of gck-1(km15) young adult hermaphrodites (L4+24 hours) revealed a significant decrease in MPK-1b activation concomitant with a striking increase in MPK-1a activation in gck-1(km15) animals (gck-1(km15) gonads suggest that loss of GCK-1 leads to germline activation of MPK-1a specifically at pachytene. However, it was previously proposed that the MPK-1b isoform is specific for germ cell development since it is only activated in animals with a functional germline, and there were no discernible germline dependent changes in the activation of the MPK-1a isoform  mutant strain produces fertile males and females, but no hermaphrodites fog-2(q71) females but not unmated fog-2(q71) females  gonads should be independent of sperm and MSP signaling. To test this, MAP kinase activation was examined in unmated fog-2(q71) females fed gck-1 or control dsRNA. DAPI staining confirmed that the gck-1(RNAi);fog-2(q71) gonads retained the gck-1(lf) phenotype, with multiple rows of small oocytes in the proximal gonad (gck-1(RNAi) females, whereas there was a clear increase in pachytene MAP kinase activation in gck-1(RNAi);fog-2(q71) animals (gck-1(RNAi);fog-2(q71) animals  phenotype should be dependent on MAP kinase. mpk-1 mutations and mpk-1(RNAi) lead to sterility characterized by defects in pachytene progression, where the “stuck” germ cell nuclei clump together and degenerate, leaving the proximal gonad devoid of nuclei gck-1(km15)/nT1 heterozygous animals by feeding mpk-1 dsRNA and their progeny were examined. All of the wt, gck-1(km15)/nT1, and gck-1(km15) hermaphrodite progeny of mpk-1(RNAi) treated mothers exhibited a strong pachytene arrest phenotype as evidenced by clumping of pachytene nuclei and proximal gonads devoid of germ cells (gck-1(km15) animals treated with, Ksr/ksr-2, Mek/mek-2 or Raf/lin-45(RNAi) (gck-1(lf) phenotype is dependent on the MAP kinase signaling pathway. Hence, GCK-1 is likely to act upstream of Raf or in a parallel pathway.A second facet of our model predicts that the 45(RNAi) . These rThe GCK-1 N-terminus harbors a predicted ERK docking site C. elegans oocytes undergo physiological cell death gck-1(lf) animals by staining live animals with Syto12, a dye that is specific for apoptotic cells gck-1(RNAi) and gck-1(km15) gonads had a statistically significant increase in apoptotic germ cells over wt controls  gonads could be due to an upregulation of the physiological germ cell death pathway or to introduction of cell damage that triggered a checkpoint-dependent apoptotic response. The C. elegans checkpoint genes him-7 and rad-5/clk-2 mediate apoptosis in response to DNA damage. Mutations in these genes decrease DNA damage-induced but not physiological germ cell death him-7(e1480) and clk-2(mn259) hermaphrodites depleted of GCK-1 was significantly higher than control treated animals (gck-1(lf) animals is not mediated by the DNA checkpoint pathway. Altogether, these results suggest that GCK-1 is an inhibitor of physiological apoptosis in the C. elegans germline.We reasoned that the increase in cell death in  animals , indicatC. elegans germinal center kinase GCK-1 is required to inhibit abnormal oocyte development and germ cell apoptosis. Furthermore, loss of GCK-1 activity results in ectopic activation of a specific MPK-1 isoform that has not previously been shown to have a significant role in the germline. An ERK docking site dependent in vitro interaction between GCK-1 and MPK-1 suggests that GCK-1 may physically sequester MPK-1 from activation. These results are in striking contrast to the reported functions of other germinal center kinase family members, the majority of which have been shown to induce MAP kinase activation and apoptosis Here we demonstrate that the C. elegans, the requirement for MAP kinase activation in this process has been difficult to assess due to the essential role of MPK-1 in pachytene progression MAP kinase activation is an essential component of oogenesis in many organisms gck-1(lf) gonads as compared to wild type C. elegans. Interestingly, LET-60/Ras gf alleles or loss of a MAP kinase inhibitor, LIP-1, also result in fewer germ cells gck-1(lf) gonads might also be attributed to inappropriately high MAP kinase activity. Conversely, GCK-1 could be affecting cell division and/or cell survival via a MAP kinase independent pathway. Indeed, other germinal center kinases, including Drosophila Slik and Hippo, play critical roles in cell cycle progression and growth control through novel signaling pathways Significantly fewer germ cells populate Deciphering the exact mechanism through which GCK-1 affects germ cell development and apoptosis will require the identification of GCK-1 binding partners and the physiological substrates of this interesting kinase. This study contributes to a growing body of evidence suggesting that the in vivo functions of the Germinal Center kinases are likely to vary in different developmental and cellular contexts. Hence the analysis of each member of this large kinase family in a variety of model organisms will be necessary to elucidate these functions and their contribution to human development and disease.C. elegans genome database predicts four alternatively spliced gck-1 isoforms . The gck-1a isoform was PCR amplified from a C. elegans cDNA library and DNA sequencing confirmed that it was identical to the predicted gck-1a cDNA (Genbank: AAC69038.1). All of the relevant experiments described here were performed with this cDNA.The C. elegans var. Bristol. The following strains were maintained at 20°C: N2 (wild-type), NL2098 (rrf-1(pk1417)I), MT1522 (ced-3(n717)IV), MT2547 (ced-4(n1162)III), CB1480 (him-7(e1480)V), SP506 (rad-5/clk-2(mn159)III), CB4801 (fog-2(q71)V), JS347 (gck-1(km15)/mIs10V) V/nT1[qIs51]). nT1[qIs51] was used as a dominant green fluorescent balancer chromosome, allowing qIs51-containing animals to be scored beginning at the four-cell stage of embryogenesis glp-4(bn2ts)I) is germline-less at 25°C and was therefore maintained at 15°C. The gck-1(km15) deletion mutant was generated by a TMP/UV method and isolated using a sib-selection protocol GAATACTTTTGTG and TATTACACTTTT, corresponding to a 1334 base pair deletion between nucleotides 13646 and 14969 in cosmid T19A5. Direct sequencing of the PCR products was used to verify the deleted region.Standard techniques were used to culture and genetically manipulate all strains gck-1a and mpk-1(RNAi) constructs, their entire coding sequences were PCR-amplified from a C. elegans cDNA library using primers corresponding to their start and stop codons with restriction sites added for subcloning. gck-1a primers: 5′-CGTATCTAGAATGACAACGACATCATCAG-3′, and 5′-GCATAAGCTTTTACCGGGGTTGTCAGTC-3′. mpk-1 primers: 5′-GTACGTCGACGCATGCCAACGTGGATACC-3′, and 5′-CTGAGCGGCCGCCTAAACAGGATTCTGCCTC-3′. For lin-45 and ksr-2, corresponding EST cDNA clones ) were PCR amplified using primers with restriction sites for subcloning. All resulting cDNAs were confirmed by sequencing, subcloned into the L4440 feeding plasmid, and transformed into HT115 (DE3) E. colimek-2/L4440 construct was obtained from the C. elegans feeding library . dsRNA production was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and fed to C. elegans hermaphrodites as previously described gck-1a resulted in a phenotype that was indistinguishable from gck-1(RNAi) by feeding.To create the All primers were synthesized by SigmaGenosys . All sequencing was performed by the M.D. Anderson DNA Analysis Core Facility , which is supported by NCI Grant CA-16672.A Leica DMR compound fluorescent microscope  equipped with a Spot digital camera  was used to capture Differential Interference Contrast (DIC) and immunofluorescent images. For DIC microscopy, animals were anesthetized in 1x anesthetic , dissected on a Poly-L-Lysine coated glass slide and covered by a 22×22 mm glass coverslip mounted on petroleum jelly.Immunofluorescent image stacks (0.2 uM per slice) were obtained using a 40X/1.3 or 60X/1.45 oil immersion lens attached to a Nikon 2000U inverted microscope equipped with a motorized x, y, and z-stage , and a CoolSnap HQ camera . Images were captured with Metamorph software  and deconvolved using AutoDeblur . Confocal image-stacks (12 slices per gonad) were obtained using a Zeiss LSM 510 confocal microscope with a 63X/1.4 oil immersion lens. Adobe Photoshop CS2  was used to prepare all figures. Within each figure, the exposure time and any post-microscopy processing  were identical for each gonad.The anti-phospho-histone H3  C. elegans germlines, adult (L4+24 hours) animals were transferred to a watch glass containing 1x anesthetic. Gonads were extruded using two 22½G-syringe needles by slicing the animals just below the pharynx. Dissected animals were transferred by mouth pipetting in 1x PBS +0.1% Tween to a glass culture tube where they were washed, and subsequently fixed in 3% formaldehyde and 0.1 M potassium phosphate for one hour, followed by post-fixation in −20°C methanol for 5 minutes as previously described For immunofluorescence analysis of For MAPK-YT immunostaining, dissections were performed as above, while fixation and MAPK-YT staining was performed as previously described Animals were dissected at L4+24 hours, fixed in 3% paraformaldehyde and 0.1 M potassium phosphate for one hour, and stained with DAPI. Image stacks were acquired and deconvolved as described above. Nuclei were counted using the manual count option in Metamorph. Nuclei counts for each region were compared to wt using Dunnett's Statistical Test using a 99% family confidence interval In wt gonads, condensed nuclei in the distal region that had a metaphase chromosome arrangement were counted as mitotic metaphase nuclei. The mitotic region as a whole was defined as nuclei residing from the distal tip up to the transition zone gck-1 loss-of-function (lf) germline nuclei were counted using the same methods, except that nuclear morphology was the only criteria used for distinguishing different stages since the temporal and spatial order of developing germ cell nuclei was severely disrupted from pachytene to diakinesis in gck-1(lf) gonads. Some nuclei did not have distinct chromosome morphology and were therefore grouped as “other.”The 100 adult (L4+24 hours) animals were individually picked into M9 solution in a microfuge tube, allowed to purge for three to five hours, and washed three times in M9. Animals were then resuspended in SDS-PAGE loading buffer E. coli expression vector (New England Biolabs (NEB), Beverly, MA). The MBP-GCK-1 fusion proteins were expressed and purified on amylose beads following manufacturer's instructions (NEB). A Glutathione-S-Transferase (GST)-MPK-1a fusion protein was created by PCR amplification of the MPK-1a open-reading frame from a C. elegans cDNA library. The MPK-1a cDNA was subcloned into the pGEX-6P-1 E. coli expression vector (GE LifeSciences). GST and GST-MPK-1a proteins were expressed and purified from E. coli using glutathione beads (GE LifeSciences) following manufacturer's instructions, except that the proteins were not eluted from the beads. The GST and GST-MPK-1a coated beads were incubated with approximately 2 ug eluted MBP-GCK-1 fusion protein in 100 ul binding buffer  for more than 2 hours at 4°C. The beads were washed four times in binding buffer, resuspended and boiled in SDS/PAGE buffer, and the bound proteins separated on a 10% Tris-glycine polyacrylamide gel. A fraction of the protein in each binding reaction was also loaded on the gel. Separated proteins were transferred to nitrocellulose and subjected to western analysis with α-MBP (NEB) and α-GST DNA sequences corresponding to the GCK-1 protein fragments in Cell death assays were performed by staining hermaphrodite animals (L4+24 hours) with Syto12 (Invitrogen) as previously described Table S1Germ nuclei counts(0.03 MB DOC)Click here for additional data file.Figure S1http://www.ebi.ac.uk/Tools/clustalw2/index.html) and BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Identical amino acids are in solid boxes and similar residues are shaded. The kinase domain is underlined and the GCK-III subfamily signature sequence is overlined. The breakpoints in the gck-1(km15) allele are indicated (*).An alignment of GCK-1 and other GCK-III subfamily protein sequences. The GCK-III subfamily was defined in . The aligned sequences are human MASK (GenBank: BAA92785.2); human MST3 (Swiss-Prot: Q9Y6E0.1); human SOK1/YSK1 (Swiss-Prot: O00506.1); Drosophila GCKIII (GenBank: AAF55388.1); and C. elegans GCK-1a (GenBank: AAC69038.1). The sequences were aligned using the ClustalW2 EMBL-EBI server: (1.03 MB TIF)Click here for additional data file.Figure S2Schematic of the C. elegans gonad. The C. elegans hermaphrodite gonad consists of two mirror image U-shaped arms. Each arm consists of a distal mitotic region (*) with proliferating germ cells. These nuclei transition into meiosis with a crescent shape characteristic of leptotene and zygotene. Pachytene nuclei are arranged on the surface of the gonad and surround a common anucleate cytoplasm, the rachis. Germ cells remain in the pachytene stage for an extended period before passing through diplotene (condensing chromosomes enclosed in a nuclear membrane) and arresting in diakinesis . In response to MSP, the oocyte most proximal to the spermatheca (S) matures: the nucleus migrates distally and nuclear envelope breakdown occurs. The mature oocyte is then ovulated through the spermatheca (S) where it is fertilized and passed into the uterus where the embryo (E) develops before being extruded into the environment.(9.19 MB TIF)Click here for additional data file.Figure S3Germ cell numbers are reduced in gck-1(lf) hermaphrodites. (A) The average total number of germline nuclei in a single gonad of the indicated genotype as grouped by nuclear stage see . (B) The(0.74 MB TIF)Click here for additional data file.Figure S4The gck-1(lf) phenotype requires the MAP kinase pathway. (A–H) DAPI stained gonads from  wt and  gck-1(km15) animals fed control , ksr-2 , mek-2 , or lin-45  dsRNA. Scale Bar, 20 Î¼m.(1.61 MB TIF)Click here for additional data file.


To assess the clinical relevance of transforming growth factor-beta1 (TGF-beta1) in hepatocellular carcinoma (HCC), urinary TGF-beta1 and serum alpha-fetoprotein (AFP) were determined in 94 patients with cirrhotic HCC, 94 age- and sex-matched patients with cirrhosis alone and 50 healthy adults. TGF-beta1 level in HCC was higher than in cirrhosis alone or in healthy controls (each P = 0.0001). There is an inverse correlation between TGF-beta1 and AFP levels . Significantly higher TGF-beta1 level was found in HCC patients with worsening Child-Pugh stages, diffuse HCC, tumour size > 3 cm, multilobular tumour and AFP < or = 20 ng ml(-1). TGF-beta1 level decreased after complete treatment with transcatheter arterial chemoembolization (P = 0.0001). The median survival in HCC patients with raised TGF-beta1 was shorter than those with normal TGF-beta1 (P = 0.018). Multivariate analysis indicated that TGF-beta1 and AFP were significantly correlated with the presence of HCC. In addition, TGF-beta1 could be used as a diagnostic marker for HCC, particularly in tumours with low AFP production. In conclusion, elevated urinary TGF-beta1 level is a tumour marker and predictor of poor survival for cirrhotic HCC.


MDM2, the genes GAGE-1, -2 coding for tumour-associated antigens and the candidate tumour suppressor gene FHIT. Different patterns of non-mutant FHIT transcripts including precise deletion of exons were found in 7/10 HCC, 2/10 FNH and 2/10 HC. However, expression of non-mutant GAGE-1, -2 RNA was demonstrated exclusively in 6/10 HCC samples. Further genetic features specific of HCC were point mutations in a zinc-finger motif of MDM2 (3/10 HCC samples). Neither GAGE-1, -2 expression nor MDM2 mutations were observed in the FNH samples, or in normal hepatocytes. Our findings suggest that occurrence of variable FHIT transcripts is not restricted to hepatic malignant tumours. In contrast, MDM2 mutations and GAGE-1, -2 expression were associated with HCC specimens. Therefore, the RT-PCR assays for GAGE-1, -2 and MDM2 might be useful adjuncts in cytodiagnosis of liver neoplasms. © 1999 Cancer Research CampaignOverexpression and/or mutations of oncogenes, tumour suppressor genes and tumour rejection genes have been observed in several human malignancies. Their analyses might be of diagnostic importance. Therefore, malignant hepatocytes derived from hepatocellular carcinoma (HCC) tissue as well as non-malignant hepatocytes derived from focal nodular hyperplasia (FNH) were studied. Samples containing normal human hepatocytes (HC) served as controls. Cellular material was obtained by fine-needle aspiration biopsy guided by ultrasound. Cells were analysed for expression and mutation of the oncogene


Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a  Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. The DNA damage signaling function of GEN-1 is separable from its role in DNA repair. Unexpectedly, GEN-1 defines a DNA damage-signaling pathway that acts in parallel to the canonical pathway mediated by CHK-1 phosphorylation and CEP-1/p53. Thus, an enzyme that can resolve Holliday junctions may directly couple a late step in DNA repair to a pathway that regulates cell cycle progression in response to DNA damage.Coordination of DNA repair with cell cycle progression and apoptosis is a central task of the DNA damage response machinery. A key intermediate of recombinational repair and meiotic recombination, first proposed in 1964, involves four-stranded DNA structures. These intermediates have to be resolved upon completion of DNA repair to allow for proper chromosome segregation. Using forward genetics, we identified a  The correct maintenance and duplication of genetic information is constantly challenged by genotoxic stress. DNA double-strand breaks (DSBs) are amongst the most deleterious lesions. DSBs can be induced by ionizing irradiation (IR) or caused by the stalling of DNA replication forks. In response to DSBs, cells activate conserved DNA damage checkpoint pathways that lead to DNA repair, to a transient cell cycle arrest, or to apoptosis and senescence. The full activation of DNA damage response pathways and DSB repair by homologous recombination (HR) depends on a series of nucleolytic processing events. Following DSB formation, broken ends are resected in a 5′ to 3′ direction to generate 3′ single-strand overhangs S. cerevisiae Cce1 and S. pombe Ydc2, both of which act in mitochondria in vitro properties of this complex currently appear somewhat imprecise, and more akin to flap endonuclease action in vitro via biochemical fractionation E. coli RuvC junction-resolving enzyme. In parallel the budding yeast GEN1 ortholog Yen1 was identified as a resolving enzyme using functional genomics based approaches. The biological functions of human GEN1 are unclear, and the deletion of yen1 has no obvious DNA repair defect yen1 in conjunction with mus81 leads to MMS hyper-sensitivity gen-1/yen1 homolog, complements the meiotic defect associated with mus81 Prototypic HJ resolving enzymes are nucleases that resolve HJs by introducing two symmetrical cleavages that result in either crossover or non-crossover products, depending on which strands are cleaved. Cuts made by junction-resolving enzymes need to be perfectly symmetrical so that products can be re-ligated, thus requiring no further processing events for HJ resolution Caenorhabditis elegans germ line as a genetic system to study DNA repair and DNA damage response pathways. As part of the C. elegans life cycle invariant embryonic cell divisions occur very rapidly. Embryonic cells tolerate a relatively high level of DNA damage using error prone polymerases, possibly a result of natural selection that favours rapid embryonic divisions at the expense of genome integrity C. elegans germ line, which is the only proliferative tissue in adult worms, displays longer cell cycles and is much more sensitive to DNA damaging agents. The gonad contains various germ cell types arranged in a distal to proximal gradient of differentiation -dependent induction of the CEP-1/p53 target EGL-1. Our results suggest that GEN-1 is a dual function protein required for the repair of DSBs as well as for DNA damage checkpoint signaling.Using unbiased genetic screening and positional cloning approaches we have cloned the C. elegans mutants hypersensitive to IR and/or defective in DNA damage-induced cell cycle arrest and apoptosis. During C. elegans development, the majority of cell divisions occur during embryogenesis. In contrast, germ cell proliferation, which commences with two germ cells at the L1 larval stage, predominates in the following three larval stages and continues in adult worms, where all somatic cells are post-mitotic, but continued germ cell proliferation results in a steady state level of ∼500 germ cells. In order to select for mutants hypersensitive to IR, worms mutagenised with ethyl methane sulphonate (EMS) were irradiated at the L1 stage with 60 Gy of IR. This dose of radiation does not overtly affect germ cell proliferation in wild type worms while mutants hypersensitive to IR display reduced levels of fertility (data not shown). Out of 906 F2 lines screened, 3 mutations  were recovered for the yp30 complementation group, each of which was derived from an independently mutagenised Po animal. In C. elegans, treatment of L4 larvae with IR leads to the activation of a DNA damage response checkpoint pathway that triggers apoptosis of meiotic pachytene stage germ cells, and a transient halt of mitotic germ cell proliferation leading to enlarged cells yp30 complementation group does not enlarge mitotic germ cells upon IR of L4 larvae, similar to the mrt-2 (e2663) checkpoint mutant (yp30 complementation group (data not shown). To show that yp30 germ cells do indeed fail to arrest cell cycle progression after irradiation, we stained N2 wild type and yp30 mutants with antibodies against phosphorylated tyrosine-15 CDK-1, which serves as a G2 marker yp30 germ cells fail to do so , but lacked the intergenic mutation found in yp30. Sequencing of the gen-1 cDNA confirmed the predicted gen-1 cDNA sequence and the predicted intron-exon structure of gen-1, available from Wormbase . A gen-1 deletion allele, gen-1(tm2940) , similarly lead to a cell cycle arrest defect upon irradiation (gen-1(tm2940)/gen-1(yp30) trans-heterozygotes (gen-1(tm2940), gen-1(yp30) and mrt-2(e2663) worms are equally defective in IR induced cell cycle arrest (gen-1(tm2940) is largely defective in DNA damage-induced apoptosis, similar to cep-1(lg12501), a deletion mutant of the C. elegans p53-like gene cep-1gen-1 is required for IR-induced apoptosis and cell cycle arrest in C. elegans germ cells.To uncover new genes involved in DNA damage response signaling, we chose an unbiased genetic approach and screened for t mutant  [39], ant mutant . We did to do so , a findito do so . To clon(tm2940)  obtainedadiation . In addiozygotes . Time coe arrest . FurtherDrosophila, and named DmGEN1 (XPG like Endonuclease-1) gen-1(yp30) mutation leads to the expression of a C-terminally truncated protein that does not affect the putative catalytic centre  strains but not for gen-1(tm2940)  .C. elegans GEN-1 exhibits Holliday junction-resolving activity in vitro, as predicted from homology to the human GEN1, recombinant wild type GEN-1, GEN-1 (yp30) and an E135A mutant were expressed and purified, the latter bearing a mutation in one of the putative nuclease active site residues  worms propagate as wild type, and fail to exhibit embryonic lethality in the absence of genotoxic stress  and gen-1(tm2940) mutants were equally hypersensitive to IR, similar to hus-1(op244), mrt-2(e2663) and hpr-17(tm1579) positive control strains (gen-1(tm2940) and gen-1(yp30) were MMS hypersensitive  and gen-1(RNAi) are as IR-sensitive as the hpr-17(tm1579) deletion, whereas gen-1(yp30) pachytene germ cells were not sensitive to IR (gen-1(yp30) allele, which results in a C-terminally truncated protein that retains nuclease activity in vitro, elicits IR- and MMS-induced hypersensitivity for mitotic germ cells of L1 larvae, whereas a null gen-1 mutation displays additional hypersensitivity to these agents in L4 germ cells arrested in pachytene. Our results suggest that the signaling function of GEN-1 is likely conferred by the C-terminus of GEN-1, given that the gen-1 (yp30) C-terminal truncation mutants as well as the gen-1 (tm2940) deletion are defective in checkpoint signaling, whereas gen-1(yp30), which retains nuclease activity that may directly promote DNA repair in mitotic germ cells. The differential sensitivity of the gen-1 (yp30) allele in L1 and L4 survival assays likely reflects the fact that checkpoint-induced cell cycle arrest contributes to the survival of mitotic germ cells to IR. Furthermore, gen-1 (yp30) is only partially defective for IR-induced germ cell apoptosis  and both gen-1 mutants is higher as compared to wild type, indicating a DSB repair defect (gen-1 mutant worms we directly assayed for chromosome fragmentation after irradiation with 90 Gy. As shown previously mrt-2(e2663) oocytes were fragmented. In contrast, IR-induced damage was repaired in wild type, where oocyte chromosomes appear as 6 morphologically intact condensed DAPI stained structures  and gen-1(yp30) are equally defective in repairing diakinesis chromosomes 48 hours after irradiation we consider it likely that the checkpoint functions of gen-1 (and mrt-2) in mitotic germ cells contribute to DSB repair. Given that DSBs inflicted in pachytene cells are repaired in gen-1 and mrt-2 mutants while this is not the case for DSBs in mitotic germ cells there might be a stronger requirement of GEN-1 and MRT-2 for DSB repair in mitotic germ cells.To test if the IR sensitivity phenotypes of r defect . To direructures . Chromosafter IR . Given tgen-1 acts in a known pathway promoting the repair of DSBs. We first examined if gen-1 affects non-homologous DNA end joining. In C. elegans DNA end joining is predominantly used in somatic cells. Worms defective in DNA end joining genes such as lig-4, cku-70 and cku-80 show a reduced pace of development upon IR of embryos gen-1 mutants exhibited any such somatic developmental delay (gen-1(tm2940) is reminiscent of the phenotype of mutations in upstream DNA damage signaling factors such as the C. elegans 9-1-1/Replication Factor C-like complex members hus-1 and mrt-2 (S. pombe rad1). Given that mutations of genes encoding for the 9-1-1 complex lead to telomere replication defects gen-1(tm2940). We failed to observe such an effect, further indicating that gen-1 is not part of the mrt-2 epistasis group  background led to an increased number of RAD-51 foci as compared to gen-1 RNAi in wild type worms and to the mrt-2(e2663) mutant in mitotic germ cells (gen-1(yp30), which is checkpoint-defective but encodes a protein that can promote HJ resolution in vitro, did not cause synthetic lethality when combined with an hpr-17 mutation, nor did it exacerbate the radiation hypersensitivity phenotype of hpr-17  deletion leads to excessive genome instability in germ cells and concomitant sterility gen-1 atl-1 double mutants. In contrast, C. elegans atm-1 plays a minor role in DNA damage signaling and atm-1(gk186) results in partial defect in IR-induced cell cycle arrest and apoptosis atm-1(gk186) deletion is not hypersensitive to IR when subjected to the L4 IR survival assay, and that the IR sensitivity is not enhanced by the gen-1(yp30) mutant (atm-1(gk186) mutant is sensitive to IR in the L1 assay, and IR sensitivity is enhanced in combination with both gen-1(tm2940) and gen-1(yp30)  mutant . In cont-1(yp30) .gen-1 might act in parallel to atm-1 for repairing mitotic germ cells affected by DNA double-stranded breaks.In summary, our results suggest that gen-1 encodes for a nuclease, we wanted to eliminate the possibility that GEN-1 might also be required for the processing of DSBs to generate single-stranded DNA overhangs, which would be coated by RPA1 and lead to the ATRIP-dependent activation of ATR in mammalian cells gen-1(tm2940) worms. We found that the sequential accumulation of RPA-1 (green) and RAD-51 (red) foci does not significantly differ between wild type and gen-1(tm2940) worms, indicating that the initial steps of DSB processing occur normally in gen-1 mutants (in vitro (Given that  mutants . These rin vitro .C. elegans ATL-1/ATR-mediated DNA damage checkpoint pathway in gen-1(tm2940) mutants, we analyzed the IR-induced transcriptional induction of the pro-apoptotic BH3-only domain encoding genes ced-13 and egl-1. The induction depends on the C. elegans CEP-1 p53-like transcription factor atl-1 (C. elegans ATR), clk-2, hus-1 and mrt-2egl-1 and ced-13 were induced to near-normal levels for tm2940 and yp30 alleles of gen-1, while no induction occurred in a cep-1(lg12501) background  mutant  mutant . This dagen-1 mutants. These defects are as severe as those observed in known C. elegans checkpoint mutants such atl-1, the worm ATR homolog clk-2C. elegans 9-1-1 complex gen-1 does not result from the ATR-, CLK-2- and 9-1-1 complex-dependent activation of the primordial worm p53-like protein CEP-1  mutation . Collectively, the absence of a meiotic defect of gen-1 together with the lack of strong synthetic effects with candidate meiotic HJ resolving enzymes, strongly suggests that C. elegans GEN-1 does not play a central role in this process.Given the specificity with which GEN-1 processes HJ structures C. elegans GEN-1 plays an essential role in responding to DSBs, but it is inert in budding yeast gen-1 mutants, likely by a combination of the above mentioned recombinational repair pathways and non-homologous end joining, but that a fraction of these breaks persists 48 hours after IR III; TG1540 gen-1(tm2940)III; TG765 cep-1(lg12501)II; TG1236 gen-1(yp30) unc-32(e189)III; TG1237 gen-1(yp30) dpy-17(e164)III; TG1233 hpr-17(tm1579)II; TG771 hus-1(op244)I; TG545 hus-1(op241)I; TG1503 hpr-17(tm1579)II: gen-1(tm2940)III; TG1502 gen-1(yp30)III, opIs76(CYB-1::YFP); TG1064 gen-1(yp42)III; TG1060 gen-1(yp45)III; TG1565 xpg-1(tm1670)I; RB964 cku-80(ok861)III; TG190 clk-2(mn159)III; VC381 atm-1(gk186)I.Worms were maintained at 20°C on NGM agar plates seeded with DNA damage-induced apoptosis and L4 radiation hypersensitivity (rad) assays were performed as described 6x-histidine tagged full length GEN-1 (pGA343) was expressed in BL21(DE3) CodonPlus cells, recovered from inclusion bodies using BugBuster (Novagen), solubilised in Urea buffer and purified with Ni-NTA following manufacturer's instructions (Qiagen). One guinea pig was immunized (BioGenes GmbH) and antibodies were affinity-purified from the final bleeding using Maltose Binding Protein (MBP) tagged protein. N- and C-terminus GEN-1 (fragments 1-136 and 356-434 respectively) tagged with MBP (pGA346 and pGA348) were purified using an amylose resin column (New England BioLabs). For affinity purification, proteins were covalently linked to AffiGel 15 (Bio-Rad).Immunostaining experiments were performed as described C. elegans full-length ORF of rpa-1 was cloned into pMAL-2c vector (New England Biolabs) and expressed in BL21 (DE3) cells. The MBP-tagged protein was purified on an amylose resin following the manufacturer's instructions (New England Biolabs) and used to immunize one rat . The same purified protein was covalently linked to AffiGel-15 (BioRad) and used to affinity-purify antibodies from the final bleed.L1 larvae stage worms were sorted from a growing population using an 11 µm filter (Millipore NY11) and treated with the indicated genotoxic agents. To test MMS and Nitrogen mustard sensitivity, worms were incubated with the indicated concentration of mutagen for 12 hours in M9 buffer. UV irradiation was performed by the XL-1000 Spectrolinker UV-C light source. 5 L1 stage worms in the P0 generation were plated onto a single plate. The number of living worms (post the L1 stage) present in the F1 generation within 48 hours of (untreated) P0 worms reaching the L4 stage was counted using a dissection microscope. For hydroxyurea (HU), L1 worms were plated on 1x NGM plates supplemented with the indicated compounds and the living adult worms corresponding to the F1 generation were established similarly. Experiments were done at least in triplicate.RNAi feeding was done as described with exception of using 1 mM IPTG 32P]-labelled on one strand in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 10 mM MgCl2, 0.1 mg/ml BSA, 0.1 mg/ml calf thymus DNA, 1 mM DTT and 1 M NDSB 201. Samples were incubated at 37°C for 30 min or overnight, and the reaction terminated by addition of EDTA. Cleavage products were analysed by electrophoresis in 12% polyacrylamide gels containing 8 M urea. Gels were dried and imaged using a Fuji BAS 1500 phosphorimager.Recombinant proteins (50 nM) were added to 2 nM  or 5 nM For the nuclease assays, substrates were generated as described Single-stranded:CGCTCTAGAGCGGCTTAGGCTTAGGCTTAGGCTTA-1971: Double-stranded :TAAGCCTAAGCCTAAGCCTAAGCCGCTCTAGAGCG-1972: 5′ Flap 3′ Flap ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC-A-Flap: CAGCAACGCAAGCTTGATGTGGAAAATCTCTAGCA-B-Flap: GGCTGCAGGTCGAC-A: CAGCAACGCAAGCTTG-B: GTCGACCTGCAGCCCAAGCTTGCGTTGCTG-C: Figure S1yp30 complementation group. (A) yp30 mutant worms fail to arrest at the G2 stage following DNA damage. Wild type and yp30 worms expressing cyclin B1 fused to YFP (gift from Michael O. Hengartner), unirradiated or irradiated (60 Gy) and assayed after 8h. (B) Representative pictures of N2-wild type and gen-1 mitotic germ lines with and without IR treatment. The black arrow depicts a small nucleus of an untreated wild type germ line. The white arrow indicates an enlarged nucleus in an IR treated wild type germ line. Numbers in each panel indicate the respective numbers of nuclei. yp42, yp45, and yp30/tm2940 trans-heterozygotes are all defective in IR induced cell cycle arrest. The number of mitotic cells is indicated in each panel.Cell cycle arrest defects of the (3.31 MB TIF)Click here for additional data file.Figure S2Quantification and time course analysis of IR dependent cell cycle arrest as described in (0.33 MB TIF)Click here for additional data file.Figure S3Caenorhabditis elegans, An, Aspergillus nidulans, Bm, Brugia malayi, Ca, Candida albicans, Ci, Ciona intestinalis, Dm, Drosophila melanogaster, Gg, Gallus gallus, Hs, Homo sapiens, Nv, Nematostella vectensis, Pp, Pichia pasteuris, Sc, Saccharomyces cerevisiae, Sp, Schizosaccharomyces pombe.Alignment of GEN-1 N (upper panel) and I domains (lower panel). Alignments were performed as described in (1.98 MB TIF)Click here for additional data file.Figure S4C. elegans GEN-1, (pGA532), GEN-1(E135A), (pGA541) and GEN-1(yp30) (pGA543) (left panel) and the human GEN1 amino acid 1-527 fragment (right panel).* indicates an nonspecific band. The arrow indicates GEN-1 while the arrowhead indicates GEN-1(yp30). GEN-1 fragments were cloned into a pGEX derivative containing a C-terminal 6-histidine tag, and induced overnight with 0.5 mM IPTG at 20°C in BL21(DE3) CodonPlus E.coli cells and purified on a cobalt column  following the manufacturers instructions. (B) Nuclease assay of human and C. elegans GEN-1 on Jbm5 junction substrate. The cleavage assay was performed at 37°C for 30 min. Respective cleavage sites are represented on the right panel. (C) GEN-1 cleaves specifically Holliday Junction structures in vitro. Holliday Junction, 5′ flap, duplex DNA, single-stranded DNA and 3′ overhang were subjected to nuclease assay.Purification of GEN-1 and in vitro nuclease assays. (A) Purification of recombinant (3.07 MB TIF)Click here for additional data file.Figure S5gen-1 (tm2940) deletion but not gen-1 (yp30) leads to DNA repair defects. (A), exposure to MMS, (B) UV irradiation, (C) exposure to Nitrogen Mustard, and (D) to exposure to HU. Assays were performed using L4 larvae as described The (1.12 MB TIF)Click here for additional data file.Figure S6gen-1 (tm2940). (A) RPA-1 (green) and RAD-51 foci (red) from wild type and gen-1 (tm2940) worms dissected for immunostaining 60 minutes after treatment (30 Gy). Scale bar is 10 µm. Statistical analysis of RPA-1 (B) and RAD-51 foci formation (C). (n = 20 cells), error bars represent s.e.m.. p-values for the comparison between wild type and mutants are between 0.27 and 0.93 indicating that there is no statistically significant difference in RPA-1 foci formation between wild type and the respective mutants.RPA-1 loading occurs in (3.37 MB TIF)Click here for additional data file.Figure S7gen-1 (tm2940) worms are wild type for a DNA end-joining assay affecting somatic cells DNA end joining assays and GEN-1 antibodies purification. (A) (0.67 MB TIF)Click here for additional data file.Figure S8gen-1 mutants and gen-1 hpr-17 synthetic lethality. (A) gen-1 (tm2940) worms do not have a mortal germ line phenotype indicative of telomere defects. Worm lines were propagated over 30 generations as described trt-1(tm899) mutant deleting the catalytic subunit of the worm telomerase was used as a positive control. (B) Confirmation of gen-1 hpr-17 synthetic lethality by double mutant analysis. 10 gen-1(tm2940); hpr-17(tm1579)/+F2 lines were selected by PCR, and F3 progeny was scored for the hpr-17(tm1579) allele. Out of a total of 36 F3 adult worms 19 were wild type, 17 were heterozygous for hpr-17(tm1579) and none was homozygous for hpr-17(tm1579). (C) gen-1(yp30) does not enhance the IR hypersensitivity of hpr-17(tm1579). Assays were performed as described in Absence of mortal germ line defect in (0.53 MB TIF)Click here for additional data file.Figure S9mrt-2(e2663) gen-1(RNAi) premeiotic germ lines. Foci were quantified 24 h post L4 (right panel). A projection of five z-stacks is shown using SoftWorXs Applied Precision 3.1 for clarity. For quantification the entire germline was projected and foci were counted (n = 5). Error bars represent s.e.m.Increased level of RAD51 foci in (2.84 MB TIF)Click here for additional data file.Figure S10gen-1 and gen-1 atm-1 double mutants by the L4 IR survival assay. Error bars represent s.e.m.Analysis of (0.49 MB TIF)Click here for additional data file.


Using Serial Analysis of Gene Expression (SAGE), comparison of gene expression profiles between growing stages animals and long-lived, developmentally diapaused dauer larvae revealed that pha-4 transcription is increased in the dauer stage.pha-4 expression by RNAi during post-embryonic development showed that PHA-4 is essential for dauer recovery, gonad and vulva development. daf-16, which encodes a FOXO transcription factor regulated by insulin/IGF-1 signaling, shows overlapping expression patterns and a loss-of-function post-embryonic phenotype similar to that of pha-4 during dauer recovery. pha-4 RNAi and daf-16 mutations have additive effects on dauer recovery, suggesting these two regulators may function in parallel pathways. Gene expression studies using RT-PCR and GFP reporters showed that pha-4 transcription is elevated under starvation, and a conserved forkhead transcription factor binding site in the second intron of pha-4 is important for the neuronal expression. The vulval transcription of lag-2, which encodes a ligand for the LIN-12/Notch lateral signaling pathway, is inhibited by pha-4 RNAi, indicating that LAG-2 functions downstream of PHA-4 in vulva development.Knocking down Analysis of PHA-4 during post-embryonic development revealed previously unsuspected functions for this important transcriptional regulator in dauer recovery, and may help explain the network of transcriptional control integrating organogenesis with the decision between growth and developmental arrest at the dauer entry and exit stages. C. elegans may arrest development at the dauer stage in response to starvation and overcrowding, but can resume development to the adult when an environment favoring growth is encountered , DR2455 daf-2(e1370III) mEx168 [rol-6(su1006) pha-4pE3::gfp], DR2458 daf-2(e1370III) mEx169 [rol-6(su1006) pha-4::gfp], DR2459 daf-2(e1370III) mEx170 [rol-6(su1006) pha-4::gfp], DR2460 daf-2(e1370III) mEx171 [rol-6(su1006) pha-4::gfp(M)], and DR2461 daf-2(e1370III) mEx172 [rol-6(su1006) pha-4::gfp(M)].Strains were maintained as described by Brenner . Strain pha-4 gene was amplified from C. elegans genomic DNA using primers 5' CGG AAT TCG TTT TAC CAC TGG CAC CAC 3' and 5' CCC AAG CTT CTG GTA TAC TCC GTT GGT G 3'. The PCR products were cloned into the RNAi vector L4440 between the EcoR I and Hind III sites. The pha-4 RNAi construct was transformed into E. coli HT115(DE3). RNAi bacteria cultivation and double-stranded RNA induction were performed as described by Kamath et al. [E. coli HT115(DE3) carrying the empty RNAi vector L4440 was fed to the same strain as controls.A 1.3-kb coding region of the h et al. . In all About ten Daf-c gravid adults were transferred to RNAi plates, and allowed to lay eggs for 4 to 6 hours before they were removed. The plates were incubated at 25°C for three days until dauer larvae formed constitutively. The dauer larvae were then transferred to fresh RNAi plates and incubated at 15°C to resume development. Animals that became L4 larvae or young adults after 3 days at 15°C were scored as recovered. Gonad and vulva morphologies of these adult animals were examined using a Zeiss Axioscope with Nomarski optics.pha-4, 5' GCG GAG CTC ATG AAC GCT CAG GAC TAT CTG 3' and 5' CGC AAG CTT TAG GTT GGC GGC CGA GTT C 3' ; rpl-21, 5' ATG ACT AAC TCC AAG GGT C 3' and 5' TCA CGC AAC AAT CTC GAA AC 3'Total RNA was extracted using the Trizol reagent (Invitrogen) following the manufacturer's instructions. Reverse transcription followed by PCR reactions were performed to determine gene transcription levels from different samples as previously described . The folpha-4 was amplified from C. elegans genomic DNA using primers 5' CAA CGA GAG GGC ATG CTG TGA AC 3' and 5' CGG GAT CCT GAT ATG GTT GGT AGT TTA ACG 3'. The PCR products were cloned into the vector pPD95.67 (a gift from Dr. Andrew Fire) between the Sph I and BamH I sites. The pha-4::gfp clone (50 ng/μl) was injected into the ovaries of daf-2(e1370) young adults along with the pRF4 rol-6(su1006) dominant transformation marker (100 ng/μl).A 5.5-kb DNA fragment that contains the 1.5-kb promoter through part of the third exon of pha-4 is functional in vivo, a 3.8-kb DNA fragment was amplified from C. elegans genomic DNA using primers 5' ACA TGC ATG CGT AAG GCA CCA GTT ATT TTC TG 3' and 5' CGG GAT CCG GCC TGC AAG AAA AAA ATT GAA AG 3'. The PCR products were cloned into the vector pPD95.67 between the Sph I and BamH I sites. The potential DAF-16 binding site (TATTTAC) was mutated to GCGGGCA using the QuickChange site-directed mutagenesis kit (Stratagene). The constructs (50 ng/μl) with normal or mutant binding sites were co-injected into the ovaries of daf-2(e1370) young adults along with the pRF4 (100 ng/μl) marker.To test whether the potential DAF-16 binding site in the second intron of It has been reported that PHA-4 function is required for exit from the dauer stage and for development of the vulva  ,46.SAGE, serial analysis of gene expression; RT-PCR, reverse transcriptase polymerase chain reaction; Daf, dauer formation; RNAi, double stranded RNAi-mediated interference; GFP, green fluorescent protein; DTC, distal tip cell.DC and DLR. designed the experiments and drafted the manuscript. DC carried out the experiments.


Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2–4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and ∼6–10% of CD3 were co-clustering with CD4 or CD8 as 70–110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200–500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3–CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation. While T cell receptors (TCR) recognize major histocompatibility complex (MHC)-associated peptide complex (MHCp) Direct molecular imaging of nano-spatial relationship between TCR/CD3 and CD4 or CD8 co-receptor on a primary T cell has not been reported. Scanning fluorescence nanoscale visualization of each of these molecules on cell-surface of CD4 or CD8 T cells is indeed lacking. Early studies of TCR/CD3-CD4 co-localization or interaction using conventional techniques such as flow cytometry, fluorescence/confocal microscopy or indirect biochemistry analyses of membrane rafts appear to be inconclusive with conflicting results in vivo clonal expansion We have recently innovated the use of near-field scanning optical microscopy NSOM  and quanAs an initial effort to directly visualize TCR/CD3 co-clustering or interaction with CD4 or CD8 co-receptor during T cell activation, we first imaged nanoscale distribution or relationship of each of these molecules on cell-surface of un-stimulated primary T cells using the NSOM/QD-based polarized imaging system. This polarized imaging system simultaneously collects two polarization components of images  or vertical  polarization component. The majority of QD-bound CD3 molecules were imaged in both 0° and 90° polarization components [yellow] and distributed as nano-clusters equivalent to two or more QD fluorescence intensity/FWHM on the cell-surface. (Aii) Two histograms show frequency distribution of fluorescence intensity (upper panel) and size (FWHM) (lower panel) of QD-bound CD3 on membrane of ten T cells. Single or multiple QD-bound CD3 were judged based on the single QD fluorescence intensity and size (FWHM) as previously published (1.73 MB DOC)Click here for additional data file.Figure S2(A) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten resting CD4 T cells. (B) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD4 on membrane of ten resting CD4 T cells. (C) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten resting CD8 T cells. (D) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD8 on membrane of ten resting CD8 T cells.(0.06 MB PPT)Click here for additional data file.Figure S3(A) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten anti-CD3 Ab-stimulated CD4 T cells. (B) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD4 on membrane of ten anti-CD3 Ab-stimulated CD4 T cells. (C) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten PMA/Ionomycin-stimulated CD4 T cells. (D) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD4 on membrane of ten PMA/Ionomycin-stimulated CD4 T cells.(0.07 MB PPT)Click here for additional data file.Figure S4Lck inhibition significantly inhibited the cluster formation of CD4(A) and CD8(B), respectively. The Lck inhibition was done by pre-treatment of T-cells with Lck inhibitor PP2 for 30 minutes. And these T-cells were stimulated with anti-CD3 antibody. Note that no significant clusters were observed for CD4 and CD8, respectively.(1.33 MB PPT)Click here for additional data file.Figure S5(A) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of anti-CD3/anti-CD28 co-stimulated CD4 T cells. (B) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD4 on membrane of anti-CD3/anti-CD28 co-stimulated CD4 T cells. (C) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten anti-CD28-stimulated CD4 T cells. (D) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD4 on membrane of ten anti-CD28-stimulated CD4 T cells.(0.06 MB PPT)Click here for additional data file.Figure S6(A) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of anti-CD3 co-stimulated CD8 T cells. (B) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD8 on membrane of anti-CD3-stimulated CD8 T cells. (C) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of ten a anti-CD3/anti-CD28 co-stimulated CD8 T cells. (D) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD8 on membrane of ten anti-CD3/anti-CD28 co-stimulated CD8 T cells.(0.06 MB PPT)Click here for additional data file.Figure S7The NSOM dual-color images show that CD28 co-stimulation or PMA/Ionomycin stimulation did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains (A) The NSOM dual-color images of one representative of the anti-CD28 Ab only-stimulated T-cells. Anti-CD28 Ab stimulation alone did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains, although it increased molecular number and density of CD3 clustering and enlarged nano-domains. Lower panels show the T cell topography and topography-fluorescence overlay images. (B) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of anti-CD28-stimulated CD8 T cells. (C) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD8 on membrane of anti-CD28-stimulated CD8 T cells. (D) The NSOMdual-color images of one representative of the PMA/Ionomycin-stimulated T-cells. PMA/Ionomycin stimulation did not induce apparent co-localized CD3 and CD8 nano- or micro-domains on the membrane of PMA/Ionomycin-stimulated CD8 T-cells. Lower panels show the T cell topography and topography-fluorescence overlay images. (E) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD3 on membrane of PMA/Ionomycin-stimulated CD8 T cells. (F) Two histograms show frequency distribution of fluorescence intensity (left panel) and size (FWHM) (right panel) of QD-bound CD8 on membrane of PMA/Ionomycin-stimulated CD8 T cells.(0.71 MB PPT)Click here for additional data file.


When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10−4 M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10−4 M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10−9 to 10−5 M) on LPS-induced TNF-α release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-α release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-α release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10


Cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors  that, in turn, regulate tumor invasion, neo-angiogenesis and inflammation. ranscript profiling of transformed human cells revealed that genes encoding wound healing, matrix remodeling and cell cycle proteins  are significantly up-regulated early after growth factor stimulation. The major inhibitor of plasmin generation, plasminogen activator inhibitor-1 (PAI-1), is among the most highly induced transcripts during the phenotypic transition initiated by TGF-β maximal expression requires EGFR signaling. PAI-1 induction occurs early in the progression of incipient epidermal squamous cell carcinoma (SCC) and is a significant indicator of poor prognosis in epithelial malignancies. Mouse modeling and molecular genetic analysis of complex systems indicates that PAI-1 regulates the temporal/spatial control of pericellular proteolysis, promotes epithelial plasticity, inhibits capillary regression and facilitates stromal invasion. Defining TGF-β1-initiated signaling events that cooperate with an activated EGFR to impact the protease-protease inhibitor balance in the tumor microenvironment is critical to the development of novel therapies for the clinical management of human cancers.Increased transforming growth factor- Elevateuman SCC, 10, 11.poptosis . In certelopment –19. The tastasis , 20–25.β1-induced,EMT-associated genes in human malignant keratinocytes [Microarray of the EMT transcriptome inseveral clinically relevant model systems has provided insights into the specificrepertoire of “plasticity” genes. Plasminogen activator inhibitor type-1, the major physiologic regulator of the pericellularplasmin-generating cascade, is a prominent member of the subset of TGF-inocytes , 26, 27.inocytes . The assinocytes –31. Incrinocytes , 32–36 ainocytes . Indeed,inocytes . Certaininocytes , 38 withinocytes . Identifinocytes –36, perhinocytes , invasioinocytes , 41.rho-ROCK signaling as required for PAI-1expression in TGF-β1-stimulatedcells. E box motifs (CACGTG) in thePAI-1 PE1/PE2 promoter regions, moreover, are platforms for a MAPkinase-directed upstream stimulatory factor (USF) subtype switch (USF-1 → USF-2)in response to growth factor addition [rho-ROCK axis impacts PAI-1 expressionthrough USF-dependent transcriptional controls. The continued definition of TGF-β1-activatedpathways that influence expression of this important target gene may lead totherapeutically useful approaches to manage human cancer. This paper,therefore, reviews data regarding the rapid transactivation of the EGFR in TGF-β1-stimulatedcells suggesting cooperativity between TGF-β1 and EGFR → MAPkinase pathways in PAI-1 gene expression.Recent findings clearly implicate EGFR/MEK/addition –44 suggeβ1 mobilizes both SMAD-dependent and -independentsignaling [β1-inducedrapid EGFR transactivation highlighting cooperativity between TGF-β1 andEGFR signaling events in vascular, epithelial, and endothelial cells. Indeed,PAI-1 induction in response to TGF-β1 is significantly attenuated by an EGFR pharmacologicinhibitor (AG1478), by molecular targeting of EGFR activity  and, more importantly, by genetic ablation of the EGFRin mouse fibroblasts [−/− cells engineered to express an EGFR construct. TGF-β1 treatment, moreover, specifically increased EGFRphosphorylation at the Y845 src-targetresidue; either mutation of thisresidue (EGFRY845F) or transfection of a DN pp60srcc- construct completely blocked TGF-β1-dependentPAI-1 induction. Similarly, TGF-β1 failed to stimulate PAI-1 expression in cultured mouseembryonic fibroblasts (MEFs) genetically deficient in three src family kinases  compared toidentically stimulated wild-type SYF+/+/+ cells. PAI-1 synthesis was restored in SYF−/−/− MEFs engineered to re-express a wild-type pp60srcc- [src kinase in the inductive response. The highly specific src family kinase inhibitor SU6656, morevover, effectively blockedTGF-β1-initiatedincreases in both pp60srcc- and EGFR phosphorylation aswell as pp60srcc- and EGFR activation . pEGFRY845 phosphorylationin response to TGF-β1 wasevident, furthermore, in wild type but not SYF−/−/− fibroblasts. TheTGF-β1-dependentformation of EGFR/pp60srcc- complexes [Y845 phosphorylation and the inhibition of TGF-β1- (butnot PDGF-) induced PAI-1 expression by the EGFRY845F mutant as wellas a DN-Src construct [srcc- interactions and, inparticular, the EGFRY845 pp60srcc- site in the kinase domain activation loop in signalpropagation [β1-initiatedSMAD2/3 activation, in contrast, was similar in both wild type and SYF−/−/− MEFs confirming that, in the contextof either EGFR or src family kinasedeficiency, SMAD2/3 activation occurs but is not sufficient for PAI-1induction. TGF-β1stimulated ERK1/2 phosphorylation in EGFR+/+ but not in EGFR−/− cells consistent with prior observations that TGF-β1-dependentERK1/2 activation is downstream of EGFR signaling [−/− MEFs, however, are fully capable of responding to exogenous TGF-β1 asSMAD2 was effectively activated  in both wild type andEGFR−/− fibroblasts [TGF-ignaling  althoughroblasts , 46, 47 pagation . The timignaling , 46. EGFβ1receptors phosphorylate SMADs downstream of growth factor engagement, itappears that the Rho/ROCK pathway modulates the duration of SMAD2/3phosphorylation [β1-initiatedSMAD2/3 activation and subcellular localization [β1-induced SMAD2 phosphorylation is not altered by EGFRblockade either pharmacologically (with AG1478), molecularly (by expression ofEGFRY721A or EGFRY845F), or by the genetic absence of EGFR[β1-stimulated PAI-1 expression in the absence of EGFRsignaling.While TGF-rylation . How Rholization , 50 is nlization , 52). TG with AG18, molecuβ1stimulates PAI-1 expression through two distinct but cooperating pathways thatinvolve EGFR/pp60srcc- → MEK/ERK signaling and EGFR-independent, butRho/ROCK-modulated, TGF-βR-directedSMAD and ERK activation [srcc-/MEK or Rho/p160ROCK) markedly reduced, andin some cases, completely inhibited PAI-1 expression. One model consistent with the available data [β1-responsivePE2 region E box in the PAI-1 promoter , while Y284 is a target site for src kinases [βRI is also subject to tyrosine phosphorylationpostreceptor accupancy [ras-raf-MEK-ERK cascade[β1 [β1-initiated signaling. TGF-β1 rapidly activates JNK through MKK4 and p38 viaMKK3/6 perhaps even in a cell type-specific fashion contributing to themechanistic complexity of pathway cross-talk. Each of these kinase systems, moreover, has been implicated in a celltype-dependency of PAI-1 gene control [The mechanism of MAP kinase activation in TGF- kinases . TGF-βRIccupancy . Suchph cascade, 47, 55.cade[β1 , 43, per control , 43, 55.β1 levels). This strategy would likely impact not onlyPAI-1 suppression but has the potential to regulate other proinvasive target genes. There is, in fact, increasing evidence that TGF-β1-induced connective tissue growth factor andfibronectin expression similarly involve EGFR/HER1 cooperative pathways . Moreover,PAI-1 repression by EGFR signaling blockade may also suppress tumorangiogenesis consistent with the well-established role of PAI-1 as an inhibitorof endothelial apoptosis and neovesselregression [β1-rich tumor microenvironment.Modulationof EGFR/HER1 signaling by specific receptor function (kinase domain) inhibitorsor neutralizing antibodies against specific EGFR1 ligands  can be an attractive therapeutic modality (particularly in the contextof neoplastic diseases associated with elevated TGF-gression . Combinagression  coupled