C. elegans. Here, we establish the transcription factor ETS-4, an ortholog of vertebrate SPDEF, as a longevity determinant. Adult worms with ets-4 mutations had a significant extension of mean life span. Restoring ETS-4 activity in the intestine, but not neurons, of ets-4 mutant worms rescued life span to wild-type levels. Using RNAi, we demonstrated that ets-4 is required post-developmentally to regulate adult life span; thus uncoupling the role of ETS-4 in aging from potential functions in worm intestinal development. Seventy ETS-4-regulated genes, identified by gene expression profiling of two distinct ets-4 alleles and analyzed by bioinformatics, were enriched for known longevity effectors that function in lipid transport, lipid metabolism, and innate immunity. Putative target genes were enriched for ones that change expression during normal aging, the majority of which are controlled by the GATA factors. Also, some ETS-4-regulated genes function downstream of the FOXO factor, DAF-16 and the insulin/IGF-1 signaling pathway. However, epistasis and phenotypic analyses indicate that ets-4 functioned in parallel to the insulin/IGF-1 receptor, daf-2 and akt-1/2 kinases. Furthermore, ets-4 required daf-16 to modulate aging, suggesting overlap in function at the level of common targets that affect life span. In conclusion, ETS-4 is a new transcriptional regulator of aging, which shares transcriptional targets with GATA and FOXO factors, suggesting that overlapping pathways direct common sets of lifespan-related genes.Aging is a complex phenotype responsive to a plethora of environmental inputs; yet only a limited number of transcriptional regulators are known to influence life span. How the downstream expression programs mediated by these factors (or others) are coordinated into common or distinct set of aging effectors is an addressable question in model organisms, such as  Caenorhabditis elegans. Our data showed that worms that lack ETS-4 lived significantly longer, revealing ETS-4′s role in the transcription network that regulates life span. We identified 70 genes whose expression was modulated by ETS-4 that function in lipid transport, lipid metabolism and innate immunity. Some of the ETS-4-regulated genes were also controlled by two other regulators of aging, the FOXO and GATA factors. We concluded that a common set of transcriptional targets orchestrate the network of physiological factors that affect aging. ETS-4 is closely related to the human ETS protein SPDEF that exhibits aberrant expression in breast and prostate tumors. Because the genetic pathways that regulate aging are well conserved in other organisms, including humans, our findings could lead to a better understanding of SPDEF function and longevity regulation in mammals.Animal life span is regulated in response to developmental and environmental inputs through coordinate changes in gene expression. Thus, longevity determinants include DNA-binding proteins that regulate gene expression by controlling transcription. Here, we explored the physiological role of the transcriptional regulator, ETS-4, in the roundworm  C. elegans a number of transcription factors function as genetic modifiers of aging, including Forkhead (DAF-16 and PHA-4) and GATA  factors The emerging picture from studies with model organisms is that animal life span is regulated by coordination of gene regulatory networks in response to environmental inputs. In C. elegans. Gene knock-out studies performed for 20 of the 26 ETS genes in mice have implicated ETS factors in diverse cellular processes such as proliferation, differentiation, migration, apoptosis, and cell-cell interactions ETS paralogs expressed in any particular cell type C. elegans, with only ten ets genes, provides a simpler and more genetically tractable model to investigate ETS factor function in vivo.In this study, we identify the ETS transcription factor, ETS-4, as a longevity determinant in C. elegans ets-4 is the apparent ortholog of vertebrate SPDEF/SAM pointed domain containing ETS transcription factor   . The mRNets-4 function, we undertook a reverse genetics approach in C. elegans and established ets-4 as a transcriptional regulator of longevity. Expression of ets-4 was observed in a number of tissues, however, transgenic rescue experiments implicated the intestine in the aging phenotype. Gene expression profiling identified ETS-4-regulated genes that function in life span determination. Strikingly, a significant number of these life span effectors have been previously shown to function downstream of the insulin/IGF-1 signaling pathway as well as the GATA factor, ELT-3. Genetic tests reveal that ets-4 functions in parallel to the insulin/IGF-1 signaling pathway, yet requires the FOXO transcription factor, daf-16, to modulate life span. Taken together, our findings identify the physiological role of ETS-4 in C. elegans and indicate transcriptional control of aging effectors by ETS-4.To decipher in vivo, ets-4(ok165) worms carrying a deletion at the ets-4 locus were analyzed. Coding sequences for both the ETS DNA binding domain, and the PNT domain, which is a protein-protein interaction domain conserved in a subset of ETS proteins, were lacking in the ets-4(ok165) worms  is a null allele for ETS-4 function.To determine the function of ETS-4 5) worms . Animalsanalysis . Therefoets-4(ok165) worms were examined for phenotypes. The larval developmental time, measured as the time taken for L1 larvae to reach the young adult stage at 20°C, was 6–8 hr longer in ets-4(ok165) than wild-type worms (ets-4(ok165) larvae exhibited apparently wild-type development and morphology. Neither arrests at particular larval stages nor molting defects were observed. The self brood size of ets-4(ok165) worms was similar to that of wild-type worms, suggesting normal fecundity (ets-4(ok165) worms laid significantly fewer eggs (109±5) than wild-type worms (142±6) (ets-4(ok165) hermaphrodites produced significantly more progeny (39±6) later in life (day 4 of egg-laying) than wild-type worms (14±2) (ets-4(ok165) males exhibited mating efficiency comparable to wild-type males in crosses with temperature-sensitive fem-3(e2006) hermaphrodites ets-4(ok165) worms exhibited a 10–13% delay in larval development and an altered egg-laying rate, but were wild-type in morphology, development and fecundity suggesting normal fitness.After outcrossing six times to the wild-type (N2) strain, the pe worms . Despiteecundity . Howeverecundity . During  (142±6) . In addis (14±2) . The etsets-4, we monitored the adult life span of ets-4(ok165) worms. Because different laboratory strains of C. elegans can have significantly different life spans ets-4(ok165) animals with those of isogenic ets-4(+) controls obtained by outcrossing ets-4(ok165) to the wild-type (N2) strain. Age-matched wild-type or ets-4(ok165) L4 stage larvae were picked and the first day of adulthood counted as day one. At 25°C, the mean adult life span of ets-4(ok165) worms (18.0±0.4 days) was significantly longer than that of isogenic ets-4(+) wild-type worms (13.3±0.6 days) (ets-4 led to a significantly longer mean adult life span compared to wild-type worms at 25°C (C. elegans longevity ets-4(ok165) animals was also monitored at 20°C. The mean adult life span of ets-4(ok165) animals at 20°C (27.1±0.8 days) was significantly longer than that of isogenic ets-4(+) wild-type worms (15.4±0.6 days), confirming the extended life span phenotype of ets-4 null mutant animals (eat) mutant animals are also slow-growing and long-lived ets-4(ok165) was examined by recording the pharyngeal pumping and defecation rates. Under well-fed conditions, the feeding behavior of ets-4(ok165) worms was indistinguishable from that of wild-type animals  . Thus, l at 25°C . Since g animals . Because animals . Thus, Eets-4 in life span regulation, we initiated ets-4 inactivation by RNAi at the L4 larval stage to bypass potential earlier developmental roles ets-4(ok165) mutant animals, ets-4(RNAi) on wild-type worms resulted in significant extension of mean adult life span  worms (ets-4(ok165) worms, we observed slow larval growth, altered progeny production and extended life span phenotypes in ets-4(uz1) mutant animals  mutant animals and enabled further use of this allele. However, ets-4(uz1) worms exhibited several unique phenotypes not observed in ets-4(ok165) worms including ruptured vulva, distorted seam cell syncytia and broken alae (data not shown). We speculate that these added effects are likely due to the interfering activity of the truncated ETS-4 encoded by the uz1 allele that would lack the ability to bind to ETS-4 target genes and yet, may interact with various protein partners through the PNT domain. Therefore, the additional phenotypes associated with the ets-4(uz1) allele may be involved with, but not necessarily restricted to, normal ETS-4 function and were not studied further. Nevertheless, analyses of two distinct deletion alleles of ets-4 enabled the identification of ets-4 loss-of-function phenotypes.We generated a second deletion allele of sequence . An abri1) worms . Similar animals . Thus, eets-4 promoter alone (Pets-4::gfp) or including the genomic DNA coding for ETS-4 (ets-4::gfp) were used to generate several independent transgenic lines. Robust GFP expression for both constructs was observed in the intestinal cells of transgenic worms starting at the 3-fold embryonic stage and was maintained through larval development and in the adult . Thus, these results expanded the observations made in previous studies using shorter regions of the ets-4 promoter in GFP constructs ets-4 expression in the intestine and neurons, key tissues known to regulate longevity in worms To better understand how ETS-4 regulates life span, we sought to identify the tissues in which ETS-4 functions. GFP reporter constructs containing 5 kb of the he adult . GFP exphe adult . Lastly,ets-4(ok165) worms by use of the gly-19 or rab-3 promoters, respectively ets-4(ok165) worms to wild-type (ets-4(ok165) worms was not affected by YFP::ETS-4 expression in neurons (ets-4(ok165) worms, indicating that the longevity and egg-laying phenotypes were separable  larvae identified 145 genes whose expression was altered with 88 genes down-regulated 2.2 fold or more (ets-4(uz1) worms, more genes (542) displayed altered expression in these animals than in ets-4(ok165) worms (ets-4(uz1) and wild-type adult worms confirmed the expression changes observed for eight out of eight genes  worms . qRT-PCRht genes .ets-4 deletion displayed similar life span extension, we predicted that the genes overlapping in these data sets would be enriched for aging effectors. A statistically significant overlap of 70 genes with altered expression in ets-4(ok165) and ets-4(uz1) worms was identified (p<0.0001) , multicellular organismal aging  and fatty acid metabolic process   . To deteC48B4.1) . Five coets-4 null worms to wild-type . Thus, the molecular signature of ETS-4, identified by gene expression profiling, substantiates a role for ets-4 in the intestine.Restoration of ETS-4 function in the intestine rescued life span of ild-type . To testild-type . This revit genes) and genes regulating fatty acid β-oxidation, such as an acyl-CoA dehydrogenase (acdh-2), an acyl-CoA oxidase (C48B4.1) and an enoyl-CoA hydratase (ech-9), were down-regulated in ets-4 mutant animals . The phospholipid (PL) and triacyglyceride (TAG) fractions from ets-4(ok165) worms visualized on TLC plates were similar to wild-type (ets-4(ok165) adult animals, were not altered compared to wild-type worms (ets-4(ok165) worms. Therefore, lipid homeostasis in ets-4(ok165) animals could likely be maintained due to the compensatory activities of other enzymes regulating fatty acid metabolism. Alternatively, lipid uptake, transport or storage in different tissues of ets-4(ok165) worms may be affected without an alteration in the total lipid levels within the whole organism.Lipid storage and metabolism have been linked to longevity regulation  animals . Thus, tild-type . The relpe worms . Thus, tDiscerning the transcriptional targets of ETS-4 enabled us to ask whether ETS-4-regulated genes change expression during the course of normal aging daf-2 and daf-16) ets-4(ok165) animals  or up-regulated (thn-1 and lys-7) in both ets-4(ok165) and daf-2(−) animals , and DAF-16 exist.To decipher whether a common set of targets exists for transcription factors that function in life span determination, we compared ETS-4-regulated genes with those that function downstream of the well-characterized insulin/IGF-1 signaling pathway. A comparison to the genes that act downstream of two components of the insulin/IGF-1 signaling pathway -proximal regions. As a first step towards testing this hypothesis, we characterized the DNA binding and transcriptional activity of ETS-4. We tested the binding of ETS-4 to ETS binding sites previously described, which display a 5′-GGAA/T-3′ core recognition sequence D∼10-9 M)  . Further∼10-9 M) . These rS. cerevisiae, LexA::ETS-4(1-345) activated transcription of a reporter gene via a promoter with LexA binding sites  upstream to 500 bp downstream of the transcription start site (TSS) of ETS-4-regulated genes. The position specific weight matrix (PWM) used to search for ETS binding motifs encompasses the sites that ETS-4 bound in vitro . Using tin vitro . Althoug animals . This isvit-5, which is one of the genes down-regulated in ets-4 mutant worms that bears conserved ETS binding sites  was shown previously to extend the mean life span of RNAi-sensitive wild-type worms Pvit-5::gfp expression was significantly reduced in the intestinal cells of ets-4(ok165) compared to wild-type worms suggesting that ETS-4 is necessary for inducing vit-5 expression  and wild-type strains. If ets-4 regulates life span by acting downstream of the insulin/IGF-1 signaling pathway, then loss of ets-4 should not significantly extend the life span of long-lived worms lacking insulin/IGF-1 signaling. Alternatively, further extension of the life span of long-lived ets-4 null mutant worms upon inhibition of insulin/IGF-1 signaling would indicate that ets-4 functions in parallel to this pathway. As previously reported daf-2(RNAi) animals were significantly long-lived (daf-2(RNAi); ets-4(ok165) animals lived longer than either ets-4(ok165) or daf-2(RNAi) worms alone (akt-1/akt-2 further extended the life span of the long-lived ets-4(ok165) worms (ets-4(ok165) animals. Because RNAi, and not null mutations, was used to inactivate signaling pathway genes, the possibility that the insulin/IGF-1 receptor pathway partially contributes to the life span phenotypes of ets-4 null mutant animals cannot be completely eliminated. However, our data support a model whereby ets-4 functions in parallel to the insulin/IGF-1 signaling pathway to modulate life span  worms . Thus, iife span .ets-4 and the insulin/IGF-1 signaling pathway by investigating if ets-4 participated in other physiological processes regulated by the pathway. Signaling through DAF-2 is critical for dauer formation. Hence, the involvement of ets-4 in dauer formation was tested first. Assaying for dauer formation at 25°C and 27°C ets-4(ok165) mutant animals compared to wild-type worms (data not shown), suggesting that ets-4 does not play a significant role in this process. Next, we tested whether ets-4 null mutant animals exhibit altered response to environmental stress stimuli since insulin/IGF-1 signaling also regulates stress resistance. To assay for heat stress response, the survival of adult ets-4(ok165) worms was monitored after a shift to 35°C. As reported previouslydaf-2(e1370) animals survived significantly longer than wild-type worms at 35°C, whereas daf-16(mgDf50) animals died faster (ets-4(ok165) and wild-type worms during the heat stress time-course  and wild-type worms during the majority of the oxidative stress time-course (ets-4(ok165) worms display wild-type response to oxidative stress. The participation of ETS-4 in the oxidative stress response pathway was also tested by assessing the genetic interaction of ets-4 with skn-1. Transcription factor SKN-1, the ortholog of mammalian Nrf proteins, is critical for oxidative stress resistance and acts in multiple longevity pathways, including the insulin/IGF-1 signaling cascade ets-4 null mutant animals, we inhibited skn-1 activity by RNAi and monitored the adult life span at 25°C. skn-1(RNAi) was previously reported to alter the expression of genes involved in oxidative stress response skn-1 by RNAi was shown to decrease the life span of daf-2 null mutant animals, but not control RNAi sensitive animals skn-1(RNAi) on ets-4(ok165) worms did not alter the extended life span phenotype of these worms  worms, when subjected to daf-16(RNAi), did not display an extended life span (daf-16 was required for the longevity phenotype of ets-4(ok165) worms suggesting that ets-4 functions upstream of, or in parallel to, daf-16 in lifespan regulation . Further, upon crossing a daf-16::gfp reporter into ets-4(ok165) worms, we did not observe an alteration in the expression of DAF-16::GFP due to loss of ets-4 (data not shown). Second, we examined whether ETS-4 antagonized DAF-16 activity by promoting its cytoplasmic retention. The function of the transcription factor DAF-16 is modulated in response to stress conditions, such as heat-shock, by altering its nuclear localization ets-4 under normal growth conditions and heat-shock (data not shown). These results suggest that ETS-4 does not regulate DAF-16 by altering its expression level or nuclear-cytoplasmic localization. Alternatively, ETS-4 and DAF-16 could function in parallel pathways to modulate distinct targets involved in longevity regulation  X strain was obtained from the C. elegans Gene Knockout Project at OMRF . ets-4(uz1) was isolated by PCR-based screening of a library of worms mutated with EMS generated at the University of Utah. The deletion breakpoints of the uz1 on cosmid F22A3 are 14790 and 15861. Single-worm PCR ets-4(uz1) and ets-4(ok165) worms. For ets-4(ok165) and ets-4(uz1) identification, nested-PCR was carried out using the following primers: Forward primers: CAATGAACGGTACTGGCTCAG and Primer P5: TGCAATCTTCCAATCCAACCC; Reverse primers: ACTGCCGGAGGACAAATGTC and Primer P6: CATTGCGATTCCCATGTAACC; Primers for sequences deleted in the ok165 and uz1 alleles: GCTAGCCAGCACCAACAATCAA and ACACCAAACGCTGCTTCTTT. The ets-4(ok165) and ets-4(uz1) worms were outcrossed to the N2 strain six times before phenotypic analysis, including life span assays. We also used BC11290: dpy-5(e907) I; sEx11290[rCesC04F6.1::GFP + pCeh361] lin-15(n765ts)daf-16(mgDf50) I, CF1041 daf-2(e1370) III E. coli expressing dsRNA from daf-16, daf-2, skn-1 . Each clone was sequenced to confirm its identity. Individual RNAi clones were grown overnight with Ampicillin (100 µg/ml) or Kanamycin (25 µg/ml) seeded onto NGM plates containing 1 mM IPTG (Sigma) and 25 µg/ml Carbenicillin (Sigma) and allowed to grow for 2 days at room temperature. For akt-1/2(RNAi), a 1:1 mix of akt-1 and akt-2 equal density overnight bacterial cultures was used to seed NGM plates containing 1 mM IPTG (Sigma) and 25 µg/ml Carbenicillin (Sigma). L4 stage larvae were placed on the RNAi plates and observed for phenotypes at 25°C.RNAi was performed essentially as described previously Taq DNA polymerase in a 50 µl reaction. Primer sequences: R1: GGCACAAGTTGTACTGATGTC, P1 (SL1 primer): GTTTAATTACCCAAG TTTGAG, P2: CAGATGACGGAGAATCAGGTC, R2: CTACAAGTTATAAGGAGGCAGG, P3: CTTCAGCCGCCTAGAAACTG and P4: CCAATATCTAGCCAGCAGGAG. The PCR product was sequenced to determine the position of SL1 attachment and splicing pattern.Total RNA from mixed stage worms was isolated by phenol-chloroform extraction and subjected to DNaseI treatment using the RNeasy kit (Qiagen). 1–2 µg of total RNA was reverse-transcribed by SuperScript III (Invitrogen) according to manufacturer's protocol. Ten percent of the resultant cDNA was PCR-amplified by ets-4(ok165) and ets-4(uz1) worms, levels of cDNA from the reverse transcription reaction were assessed by quantitative PCR according to manufacturer's protocol using the Roche LightCycler 480. Transcript levels were normalized to the averaged levels of cdc-42 and pmp-3To assess the expression of mRNA in synchronized populations of N2, C. elegans 22K gene expression arrays (Agilent). Data from three independent repeats of the experiment were analyzed. Lowess-normalized log (base 10) ratios of mutant/wild-type gene expression were obtained from the two-color Agilent C. elegans gene expression microarrays. The log ratios were analyzed using the Rank Products method For microarray analysis, total RNA was isolated from L4 stage larvae as described above and reverse transcribed. Cy5 and Cy3 labeled cDNA was applied to et al., 2002) and are accessible through GEO Series accession number GSE21851 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21851).The microarray data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar ets-4(ok165) and ets-4(uz1) worms were outcrossed to the wild-type strain six times before the life span assays. Briefly, worms were grown for two or more generations at the assay temperature (25°C or 20°C) prior to the assay. Hermaphrodites were allowed to lay eggs for 6–8 hr on OP50 to obtain synchronous progeny for the experiment. L4 stage larvae were picked to plates spotted with RNAi bacteria containing 1 mM IPTG or OP50 bacteria and allowed to age at 25°C. The animals were moved to fresh plates daily during the reproductive period and every other day for the rest of the assay. The worms were scored for life by assessing movement to touch every 1–2 days Life span assays were conducted as described previously Single L4 stage larvae were allowed to lay eggs at 20°C and transferred to a fresh NGM plate every day till the end of the reproductive period. The number of eggs laid and the number of hatched progeny were counted. The average number of eggs laid each day during the egg-laying period and the total number of progeny per worm (brood size) were plotted. Development time assays were done as described previously Figure S1H. sapiens (gray) and 10 C. elegans ETS domains (Ce) were aligned using ClustalW (version 1.83). DRAWGRAM (from phylip version 3.66) was used to construct a dendrogram of the aligned sequences. The horizontal length of the branches predicts the evolutionary distance between the genes.Phylogenetic analysis of ETS domain sequences. The protein sequences of 27 (9.87 MB TIF)Click here for additional data file.Figure S2ets-4 mutations do not alter feeding rate or defecation rate. (A) Pharyngeal pumping rates (pumps/minute) were counted for wild-type (WT) (n = 11), ets-4(ok165) (n = 10) and ets-4(uz1) (n = 12) worms (mean ± SEM). (B) Mean defecation cycle periods (seconds/cycle) of wild-type (WT), ets-4(ok165) and ets-4(uz1) worms (mean ± SEM). At least 10 worms per genotype were observed for 10 cycles each.(3.76 MB TIF)Click here for additional data file.Figure S3ets-4(ok165) animals. (A) The number of eggs laid (mean ± SEM) during each day of the egg-laying period. The egg-laying rate of wild-type (WT) worms, ets-4(ok165) Control and that of strains with tissue-specific expression of ets-4 in intestinal cells, ets-4(ok165);[Pgly-19::yfp::ets-4], or neurons, ets-4(ok165);[Prab-3::yfp::ets-4] at 20°C. * indicates p<0.001, comparing ets-4(ok165) transgenic lines to WT. (B) Total number of progeny (brood size) was counted for the genotypes indicated at 20°C. The average brood size (mean ± SEM) of ets-4(ok165) Control, ets-4(ok165);[Pgly-19::yfp::ets-4] and ets-4(ok165);[Prab-3::yfp::ets-4] are indicated.Tissue-specific expression of ETS-4 does not affect the egg-laying rate or brood size of (7.80 MB TIF)Click here for additional data file.Figure S4ets-4(ok165), ets-4(uz1) and wild-type worms. Gene expression quantified by real-time PCR in ets-4(ok165) and ets-4(uz1) mutant worms relative to wild-type (WT). Error bars represent standard error. (A) Expression of genes significantly down-regulated in ets-4(ok165) worms  and ets-4(uz1) worms  relative to WT. (B) Expression of genes significantly up-regulated in ets-4(ok165) and ets-4(uz1) worms (shown are lys-7 and thn-1) relative to WT.Expression levels of selected genes in (5.96 MB TIF)Click here for additional data file.Figure S5ets-4(ok165) worms. (A) Relative abundance of triacylglycerides and phospholipids expressed as percentage of total fatty acids (±standard error) as determined by gas chromatography. (B) Relative abundance of selected fatty acid species expressed as percentage of total fatty acids (± standard error) of WT and ets-4(ok165) worms as determined by gas chromatography. (C) TLC analysis of lipids extracted from wild-type (WT) (lane 1) and ets-4(ok165) animals (replicates in lanes 2 and 3). TAG, triacylglycerides; PL, phospholipids.Lipid levels and fatty acid composition is unaltered in (9.58 MB TIF)Click here for additional data file.Figure S6S. cerevisiae. An yeast strain that contains an integrated LacZ reporter with 8 LexA binding sites was transformed with the indicated LexA::ETS-4 fusions. Cells lysates were subjected to a colorimetric assay to assess β-galactosidase activity (LacZ activity). The relative LacZ activity was calculated by normalizing the LacZ values to that of control strains transformed with the LexA DNA-binding domain. The expression levels of the LexA proteins were comparable (data not shown). Inset is a schematic of ETS-4. (B) Transient expression assays performed in NIH3T3 cells. Luciferase activity was measured from cells transfected with a GAL4-dependent luciferase reporter and expression vectors for GAL4 DNA-binding domain alone or as a fusion to ETS-4. Relative luciferase activity (RLA) was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity (mean± SEM). The expression levels of the GAL4DBD proteins were comparable (data not shown).Transcriptional activity of ETS-4. (A) Transcriptional activity of ETS-4 in (4.44 MB TIF)Click here for additional data file.Figure S7Pvit-5::gfp in ets-4(ok165) worms. (A) Quantification of Pvit-5::gfp expression in synchronized 1-day old wild-type (WT) and ets-4(ok165) adult hermaphrodites using ImageJ (version 1.36b) software. The mean fluorescence (±SEM) of ets-4(ok165)[Pvit-5::gfp] worms was measured in a fixed area at the start of the intestine and compared to that of Pvit-5::gfp worms in an identical area. (B) Representative example of the reduced Pvit-5::gfp expression in ets-4(ok165) worms compared to wild-type (WT) worms.Reduced expression of (4.60 MB TIF)Click here for additional data file.Figure S8ets-4(ok165) animals and genetic interaction between ets-4 and skn-1 for life span. (A) Survival kinetics of animals under heat stress conditions (35°C). Fraction of animals alive at different time points (hours) after a shift to 35°C (mean ± SEM) is plotted. * indicates p<0.05, compared to WT. (B) Survival kinetics of animals in the presence of paraquat (oxidative stress conditions - See ets-4(ok165) worms grown at 25°C and subjected to skn-1(RNAi) or an empty vector (EV) control RNAi starting at the L4 stage. See Stress resistance of (10.00 MB TIF)Click here for additional data file.Table S1ets-4(ok165) and ets-4(uz1) Mutant Worms.Summary of Data from Repeat Trials of Life Span Analysis for (0.11 MB DOC)Click here for additional data file.Table S2ets-4(ok165) Compared to Wild-type (WT) Worms.Genes with Altered Expression in (0.09 MB DOC)Click here for additional data file.Table S3ets-4(uz1) compared to Wild-type (WT) Worms.Genes with Altered Expression in (0.26 MB DOC)Click here for additional data file.Table S4ets-4(ok165) and ets-4(uz1) Worms Relative to Wild-type Worms.Genes with Altered Expression in (0.10 MB DOC)Click here for additional data file.Table S5Position of Conserved ETS Binding Motifs in the Transcriptional Control Regions of ETS-4-Regulated Genes.(0.17 MB DOC)Click here for additional data file.Table S6Oligonucleotide primers used in RT-PCR analyses.(0.04 MB DOC)Click here for additional data file.Text S1Supplemental (0.07 MB DOC)Click here for additional data file.


PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.The  C. elegans, DAF-2 signals to the PI3 kinase (PI3K) homolog AGE-1. AGE-1 generates phosphatidyl inositol 3, 4, 5 triphosphate, or PIP3, which is in turn responsible for the activation of the AKT-1, AKT-2 and SGK-1 serine threonine kinases in a PDK-1 dependent manner C. elegans orthologue of the human tumor suppressor gene PTEN. Like PTEN, daf-18 encodes a PIP3 phosphatase that antagonizes the activity of AGE-1 in the DAF-2 pathway to regulate lifespan daf-18 mutations suppress the lifespan phenotype of daf-2 and age-1 mutants, it has been proposed that the role of DAF-18 in lifespan regulation relies on inhibition of the insulin/IGF-1 signaling pathway .The insulin/IGF-1 DAF-2 receptor pathway is implicated in lifespan control in several species daf-18 dependent manner, focusing on potential protein kinases. We identified one clone encoding the protein kinase SMG-1. Genetic analyses strongly suggest that SMG-1 acts in parallel to DAF-2, AGE-1 and AKT-1, but requires DAF-16, to modulate lifespan. SMG-1 is conserved across species and is involved in a mechanism responsible for the degradation of premature stop codon containing mRNA, also called NMD for “nonsense mediated mRNA decay” in C. elegans and in mammalian cells p53 C. elegans ortholog, cep-1, daf-18, and daf-16. Furthermore, our results uncover a role for SMG-1 in oxidative stress response that may be responsible for its effect on lifespan. Overall, our study unveils a novel role for SMG-1 in oxidative stress response and lifespan regulation that may be conserved in mammals.In order to identify new DAF-18 partners, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan in a rrf-3 (pk1426) single and rrf-3 (pk1426); daf-18 (e1375) double mutants. The daf-18 (e1375) mutation is a hypomorphic allele of daf-18, while the rrf-3 mutant was chosen because it shows an enhanced sensitivity to RNAi feeding daf-2, age-1 and akt-1, resulted in a daf-18 dependent lengthening of lifespan, validating our experimental approach.The screen was performed with smg-1 gene. The average lifespan of the rrf-3 strain was increased by 25% when worms were fed either one of the two non-overlapping RNAi clones for smg-1 (C48B6.6 and C48B6.7) and the increase in lifespan was completely suppressed in rrf-3(pk1426); daf-18(e1375) mutants  null allele smg-1(r861) mutant animals was not increased compared to wild-type animals (data not shown). Nonetheless, despite the fact that these mutants did not live longer than wild-type, we observed a delayed accumulation of the aging marker lipofuscine smg-1(r861) mutants, as observed in smg-1 RNAi treated animals (data not shown).We next tested whether the previously isolated smg-1(r861) null mutants are associated with a fully penetrant protruding vulva phenotype , while this phenotype was only observed in 31% (n = 969) of smg-1 RNAi treated animals. The majority of other smg-1 mutants we tested behave like the smg-1(r861) null allele (data not shown) besides smg-1(tm869) mutants (recently isolated by the Japanese C.elegans knockout consortium). An exception is the smg-1(tm869) allele, which results in a protruding vulva phenotype with similar penetrance  to smg-1 RNAi fed animals. Indeed, lifespan tests revealed that the smg-1(tm869) mutation increases average lifespan by more than 20%  or daf-2(e1370); rrf-3 (pk1426) double mutants were maintained at the semi-nonpermissive temperature 22°C smg-1 RNAi treated worms had reached the adult stage after 4 days, compared to 68+/−4.8% of worms grown on HT115 control bacteria (P = 0.02). These data strongly suggest that SMG-1 is not a broad positive regulator of the insulin pathway.The DAF-2 pathway is also critical for controlling dauer formation. To further assess a potential functional link between SMG-1 and DAF-2 we tested the involvement of SMG-1 in dauer formation. One would predict that if the role of SMG-1 in lifespan control relies on its interaction with the DAF-2 pathway, then SMG-1 inhibition should increase dauer formation. However, we found that Overall our data support a model in which SMG-1 functions at least in part independently of DAF-2 to regulate lifespan.smg-1 encodes a conserved serine threonine kinase involved in nonsense mediated mRNA decay (NMD), a mechanism responsible for the degradation of mRNAs containing a premature stop codon C. elegans and in mammalian cells. Genetic studies have determined that smg genes are regulators of the phosphorylation state of SMG-2. SMG-1, SMG-3 and SMG-4 are required for the phosphorylation of SMG-2, whereas SMG-5, SMG-6 and SMG-7 are involved in its dephosporylation In addition to SMG-1, six evolutionary conserved SMG proteins are also required for NMD in smg genes should also have an effect on lifespan. The average lifespan of worms fed with smg-2, smg-4, smg-5 or smg-7 RNAi clones was not significantly increased  inactivation is sufficient to suppress smg-1 lifespan regulation without completely inhibiting the smg-1 stress resistance phenotype.Overall, our results show that positive and negative regulators of SMG-1 activity in lifespan regulation act in a similar manner in the oxidative stress response. These data support the hypothesis that the resistance to oxidative stress of smg-1 in the regulation of lifespan via sensory neurons. Firstly, among the different mechanisms that modulate C. elegans lifespan, mutations in sensory neurons lengthen lifespan in a daf-16 dependent manner rrf-3 RNAi hyper-sensitive background smg-1 RNAi increased lifespan in RNAi hyper-sensitive backgrounds such as rrf-3 and ppw-1Several observations prompted us to investigate the role of smg-1 genetically interacts with tax-4 and daf-19 , two genes involved in lifespan regulation by sensory neurons tax-4(p678); rrf-3(pk1426) double mutants was increased when these animals were fed with smg-1 RNAi ; daf-19(m86) double mutants daf-18 RNAi  mutants, and in daf-19(m86); daf-12(sa204) mutants. Therefore, while daf-19(m86) mutants do not stain at all daf-19 expression by RNAi is not strong enough to interfere with sensory neurons development, as also observed by others . Furthermore, smg-1 RNAi treated animals stained a similar number of sensory neurons as animals fed on control bacteria . Overall our results show that smg-1 inhibition by RNAi does not compromise sensory neurons formation. These results do not however exclude a possible function for SMG-1 in sensory perception. The smg-1 predicted promoter contains the canonical DAF-19 target sequence smg-1 expression may be regulated by DAF-19. As daf-19 expression is restricted to sensory neurons, smg-1 may well play a role in these cells to regulate lifespan. Nonetheless, the SMG-1 mode of action differs from the previously described pathways, as sensory mutants, but not smg-1 inactivation, affect DAF-16 nuclear translocation smg-1 inactivation, resistance to oxidative stress was increased after daf-19 RNAi, and was suppressed when daf-19 was inactivated in combination with daf-16 or daf-18  IV daf-18 (e1375) IV; rrf-3 (pk1426) II, rrf-3(pk1426) males were crossed to daf-18(e1375) hermaphrodites and F2 progeny were assayed for sterility at 25°C and incapacity to form dauer on overgrown plates. Double mutants tax-4(p678) III; rrf-3(pk1426) II were generated by crossing tax-4(p678) III hermaphrodites to rrf-3(pk1426) II males. F2 progeny were screened for sterility at 25°C and dauer constitutive formation at 27°C. To obtain single mutants unc-54(r293) I, N2 males were mated to smg-3(r930) IV; unc-54(r293) I and paralyzed F2 were isolated. To generate rrf-3(pk1426) mutants carrying the DAF-16∶∶GFP transgene, rrf-3(pk1426) males were crossed to TJ356 To construct double mutants smg-1), F46B6.3 (smg-4), W02D3.8 (smg-5), Y43B6A.a (smg-7), B0334.8 (age-1), C12D8.10 (akt-1), R13H8.1 (daf-16), F52B5.4 (cep-1), F33H1.1 (daf-19) and T07A9.6 (daf-18) have been purchased from. daf-2 and smg-2 clones were kindly provided by C. Kenyon lab  and D. Longman  respectively. Each clone has been sequenced to confirm its identity.Bacterial feeding RNAi experiments were carried out essentially as described previously Double RNAi experiments were carried out by mixing the bacterial cultures directly before seeding the NGM plates. Controls were RNAi clone 50% diluted with vector control RNAi bacteria.smg-1 RNAi was also performed in absence of 5-FU and gave similar results (data not shown).Animals were grown on regular NGM plates at 20°C until reaching the L4 stage and then transferred to RNAi plates (F0). F0 adults were removed after 24h and F1 L4 were transferred to 10 µM 5-fluorodeoxyuracile  containing plates to prevent growth of progeny. Lifespan assays were performed at 20°C. The day of the shift is counted as day 0 in the adult lifespan assay. Control and experimental animals were transferred in parallel to fresh RNAi plates once a week. Lifespan was assessed every 2–3 days and animals were scored as dead when they ceased moving and responding to prodding. Animals that crawled off the plate, had a “protruding vulva” or an “exploded vulva” phenotype were censored. Survival analyses were performed using the Kaplan Meier method and the significance of differences between survival curves calculated using the log rank test. The statistical software used was SPSS, Version 11.5  and all P-values<0.05 were considered significant.smg-1 RNAi clone mixed either with the control, or daf-18, or daf-16 RNAi clones. F1 animals were scored for GFP expression at the L4 stage. For GFP intensity quantification, animals were photographed under a GFP filter and the average brightness was determined for each photograph by Lucia Nikon software. All images were handled identically. At least 30 animals per RNAi conditions were averaged.Animals carrying the PTC transgenic reporter P-values were calculated using the t-Student test to determine differences in oxidative stress resistance.Synchronously cultured animals were kept on NGM plates at 20°C until the young adult stage. For each strain, 5 × 20 young adults were transferred on Paraquat  containing plates . Surviving animals were scored every day during 8–9 days. At least three independent RNAi experiments have been conducted for each clone tested. daf-2(e1370) ; rrf-3(pk1426) double mutants were fed at 22°C with either HT115 or smg-1 RNAi bacteria, then removed 24 hours later. F1 progenies were followed every day and the numbers of worms that have reached the adult stage were counted at day 4. Numbers are given for 3 independent experiments.Five young adults smg-1(r861) mutant background and after smg-1 RNAi inactivation in a rrf-3(pk1426) mutant background by fluorescence microscopy under a GFP filter. About 10 worms were mounted on agar pads (2% agarose with 5 mM tetramisole) to avoid DAF-16∶∶GFP translocation due to stress The sub-cellular localization of the DAF-16∶∶GFP protein was analyzed in the Sensory neurons were stained by incubating L4 worms in M9 containing DiO (Molecular Probes) at 10 µg/ml final concentration for two hours. Worms were then transfered to plates for one hour and observed by fluorescence microscopy under a GFP filter.Table S1List of clones tested in the screen.(0.03 MB XLS)Click here for additional data file.Table S2Lifespan data for individual experiments(0.33 MB DOC)Click here for additional data file.


Correction for:Saccharomyces cerevisiae. PLoS Biol 5(12): e324. doi:10.1371/journal.pbio.0050324Buhler C, Borde V, Lichten M (2007) Mapping meiotic single-strand dna reveals a new landscape of DNA double-strand breaks in Two figure legends refer to the wrong strain of yeast.rad50S (MJL2404) were determined from input samples and immunoprecipitates (ChIP).In Figure 2, the second sentence of (B) should say: Spo11 enrichment ratios for rad50S (MJL2404); blue, BND cellulose-enriched ssDNA from dmc1Δ (MJL3095); red, BND cellulose-enriched ssDNA from spo11Y135F dmc1Δ (MJL 3096).In Figure 3, the first sentence should say: In each plot, normalized, unsmoothed array signals (average of two experiments) are as follows: orange, Spo11 ChIP from


The βla-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. βla-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of βla-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of βla increased the sensitivity of our βla-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a β-lactamase-Nipah Matrix (βla-M) fusion protein. We first codon-optimized the bacterial β The henipaviruses, Nipah (NiV) and Hendra (HeV), are emerging zoonoses; the former caused multiple outbreaks of fatal encephalitis in Malaysia, Bangladesh, and India with mortalities ranging from 40-70% while the latter produced respiratory syndromes among thoroughbred horses in Australia whilst also being implicated in the death of a horse handler -4. TheseRhabdoviridae rather than the pleomorphic Paramyxoviridae. Therefore, neither assay may fully recapitulate the biological properties of native envelope structures of live henipaviruses. Moreover, pseudotype reporter virus assays depend on efficient transcription and translation of a reporter gene after virus entry. Thus, earlier steps in viral entry, such as matrix uncoating, may also not be resolved by either of these assays.BSL-4 containment limits the opportunities for thorough analysis of live henipavirus entry characteristics. Surrogate assays to study henipavirus entry at less than BSL-4 conditions exist, such as cell-cell fusion or VSV-based NiV-envelope pseudotyped reporter assays. These assays have been used to probe envelope receptor interactions and characterize the determinants of fusion with regards to both the fusion (F) and attachment (G) envelope glycoproteins -10. Howee.g. Sendai, HPIV-1, Ebola, HIV, Rabies) or only in combination with envelope proteins  . Fig. Figonlycat/Km) for cefazolin by 1.5-fold[Y105W-M) in order to increase the assay sensitivity and make the system more amenable to high-throughput tasks. Indeed, βlaY105W-M increased the signal to noise ratio obtained in our VLP entry assay 1.8-fold  via detection of viral matrix protein exposure to the cellular cytoplasm.Our βla-M VLPs adopt the pleomorphic morphology of paramyxoviruses and incorporate henipaviral envelopes in a manner indistinguishable from wild-type NiV-M VLPs. NiV and HeV envelope bearing βla-M VLPs recapitulate their biological phenotypes in terms of receptor usage and the requirements for F and G in the paramyxoviral entry process Figs. , 4 and 5Rhabdoviridae can functionally accommodate many different heterologous envelopes [While it is clear that nvelopes -54, it invelopes -46 . GeneArt  performed mammalian codon-optimization of the NiV-M gene (M) product according to in-house proprietary software that addresses codon usage, elimination of cryptic splicing sites, as well as the stability of DNA/RNA secondary structures. NiV-M was subcloned into pcDNA3.1  between HindIII and XhoI restriction enzyme sites. The sequence of the codon-optimized NiV-M has been deposited into GenBank (Accession: EU480491). Origene  codon-optimized the βla gene, which was then subcloned into a pVAX1 (Invitrogen) expression vector between the KpnI and XhoI restriction enzyme sites. The sequence of the mammalian codon-optimized βla has been deposited into GenBank (Accession: EU744548). The βla gene was fused upstream of the NiV-M gene by overlap PCR and subsequently cloned into pcDNA3.1 via flanking KpnI and XhoI restriction enzyme sites with a NotI restriction enzyme site engineered in between the two genes. A single Y105W amino acid mutation within the βla active site was introduced using site-directed mutagenesis with QuikChange™ . βlaY105W was then cloned into pcDNA3.1 via flanking KpnI and NotI restriction enzyme sites. All gene products were confirmed by sequencing.The codon-optimized NiV-F or HeV-F and NiV-G or HeV-G gene products were tagged at their C-termini with an AU1 or hemagglutinin (HA) tag, respectively, as previously described,39. NiV-st Century Biochemicals, Marlboro, MA). Monoclonal anti-HeV specific antibodies were produced by expressing HeV-F, HeV-G, and NiV-M in rabbits then isolating and screening specific anti-HeV lymphocytes from rabbit spleens as previously described for anti-NiV-F specific monoclonal antibodies[Production protocols to provide polyclonal antibodies  via immunized rabbits (using a 20-mer antigenic peptide sequence corresponding to amino acids 29-49 of NiV-M) were generated by the Pinnacle Antibody Program (21ntibodies.293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (FBS) . Vero cells were grown in minimal essential medium alpha (Invitrogen), containing 10% FBS. CHO stable cell lines expressing ephrinB2 or ephrinB3 were derived and maintained as previously described[la-M expression plasmids (25 μg) and either NiV-F and G or HeV-F and G (10 μg each) or pcDNA3 (20 μg) expression plasmids were transfected into 10 cm dishes of 293T cells using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, supernatants were collected and clarified before pelleting the VLPs at 110,000 g through a 20% sucrose (in PBS) cushion followed by resuspension in PBS (Invitrogen) containing 5% sucrose.ββla-M VLP-containing supernatants were lysed and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently detected by immunoblotting using rabbit-anti-NiV-matrix , goat-anti-HA-HRP  , or mouse-anti-AU1   antibodies. Primary and secondary antibodies were used at 1:1,000 and 1:80,000 dilutions, respectively, or 1:10,000 for anti-HA-HRP followed by FEMTO  detection. Due to the similar molecular weights of βla-M (~70 kDa) and NiV-G (~67 kDa), membranes were probed for NiV-M, NiV-F or HeV-F, and NiV-G or HeV-G individually.200-mesh Formvar carbon-coated copper grids  were floated on drops of the NiV VLP suspensions at room temperature, then blotted and stained with 1% aqueous uranyl acetate (UA) for NiV VLPs and 2% aqueous solution of phosphotungstic acid (PTA) for VSV particles. Electron microscopy studies were performed on a Philips 201 electron microscope at 70 kV.g) with βla-M VLPs for 2 h at 37°C. Although not required for efficient VLP entry, spinoculation has been shown to significantly improve the entry efficiency of several viruses  into target cells[Cells were plated into 24-well plates at a confluency of 75% and spinoculated , beta-lactamase (bacteria) and average Homo sapiens genes.Click here for file


Identifying the molecular mechanisms and neural circuits that control learning and memory are major challenges in neuroscience. Mammalian MAGI/S-SCAM is a multi-PDZ domain synaptic scaffolding protein that interacts with a number of postsynaptic signaling proteins and is thereby thought to regulate synaptic plasticity C. elegans nematodes carrying a mutation in the single MAGI ortholog magi-1, we have identified specific neurons that require MAGI-1 function for different aspects of associative learning and memory. Various sensory stimuli and a food deprivation signal are associated in RIA interneurons during learning, while additional expression of MAGI-1 in glutamatergic AVA, AVD and possibly AVE interneurons is required for efficient memory consolidation, i.e. the ability to retain the conditioned changes in behavior over time. During associative learning, MAGI-1 in RIA neurons controls in a cell non-autonomous fashion the dynamic remodeling of AVA, AVD and AVE synapses containing the ionotropic glutamate receptor (iGluR) GLR-1 While investigating the behavioral defects of C. elegans are likely carried out by distinct subsets of interneurons. The synaptic scaffolding protein MAGI-1 plays a critical role in these processes in part by regulating the clustering of iGluRs at synapses.Together, these results indicate that different aspects of associative learning and memory in  Mammalian MAGI/S-SCAM is a postsynaptic multi-PDZ domain-containing MAGUK with a unique inverted domain organization Dynamic changes involving the formation of new synapses, morphological changes of dendrites, increased numbers of dendritic spines and the redistribution of synaptic proteins create the remarkable plasticity of the nervous system C. elegans composed of 302 neurons allows non-associative (adaptation and habituation) as well as associative learning between a variety of volatile or soluble chemoattractants, or cultivation temperature, and food. In order to investigate associative learning in C. elegans, several learning assays have been established, for example, salt chemotaxis learning C. elegansC. elegansC. elegans can provide important insights into the mechanisms of learning and memory in vertebrates including humans.The relatively simple nervous system of C. elegans. Therefore, C. elegans is an ideal model to study the function of the MAGI-1 protein in associative learning and neural plasticity. In this study, we generated a deletion mutant of magi-1 and investigated the learning and memory defect in the mutant worms. We found that loss of magi-1 function, regardless of the sensory input, impairs associative learning in C. elegans. Expression of the wild-type MAGI-1 protein in a single pair of interneurons can rescue the learning defects. In addition, magi-1 mutant worms show defects in memory consolidation, which requires the wild-type MAGI-1 protein in a distinct set of interneurons. We also found that MAGI -1 controls the distribution of ionotropic glutamate receptor, GLR-1 during learning and memory consolidation. We propose a molecular mechanism for MAGI-1 during neural plasticity, which modulatory function may be conserved in mammals.Vertebrate genomes encode three MAGI isoforms, while a single MAGI-1 ortholog exists in zh66) of the single C. elegans MAGI ortholog magi-1  deletion allele removes most of the region common to both isoforms. Total protein extracts from zh66 animals lacked both isoforms as determined by Western blot analysis (zh66 deletion likely represents a magi-1 loss-of-function (lf) allele. magi-1(lf) mutants appear healthy, fertile and display no obvious morphological or locomotory defects (data not shown).In order to study MAGI function in a simple animal model, we generated a knockout allele  animals towards different compounds. The chemotaxis of magi-1(lf) mutants to six different volatile attractants, one soluble attractant, and a repellent was comparable to the response of the wild-type N2 strain (magi-1(lf) mutants responded similarly to food starvation, indicating that magi-1(lf) mutants have no obvious sensory or motor defects (magi-1(lf) animals both exhibited strong chemotaxis towards diacetyl (DA) (magi-1(lf) mutants exhibited only a partial reduction in chemotaxis towards DA (magi-1(tm446) deletion that removes only the long isoform, on the other hand had no significant effect on olfactory learning (magi-1(lf) mutants was due to changes in adaptation rather than association, we conditioned magi-1(lf) mutants and wild type controls with DA in the presence of abundant food (magi-1(lf) mutants were not defective in DA adaptation when compared to wild-type  . Howeverwards DA . We obtalearning . This reant food . In thisild-type .magi-1(lf) mutants also showed strong defects in gustatory  and thermotaxis conditioning assays. Starvation of wild-type animals in the presence of NaCl induced a strong avoidance of NaCl in subsequent chemotaxis assays (magi-1(lf) mutants, on the other hand, showed a greatly reduced avoidance of NaCl after starvation conditioning (magi-1(lf) animals failed to associate low (15°) or high (25°) temperatures with food starvation  mutants  mutants, while no rescue was observed with Pglr-1-driven magi-1::gfp  mutants in an immediate recall test. To further confirm that RIA plays a central role in associative learning, we ablated the RIA neuron using glr-3 promoter-driven activated ICE caspase  mutants  mutants, magi-1(lf) mutants showed a defect in their nose-touch response that could be rescued by expressing magi-1::gfp under the control of the glr-1 promoter  mutants. In magi-1(lf) mutants the size of the GLR-1::GFP clusters was already enlarged in naïve animals and did not decrease after starvation conditioning (magi-1(lf) worms carrying the Pglr-1::magi-1, the Pglr-6::magi-1 or both transgenes together. Pglr-1::magi-1 alone reduced the size of GLR-1::GFP clusters to the size observed in naïve wild-type animals, but cluster size did not decrease further upon starvation conditioning . However, GLR-1::GFP cluster size in naïve Pglr-6::magi-1 animals was still increased similar to non-transgenic magi-1(lf) mutants (magi-1(lf): 2.328±0.19 µm n = 85; magi-1(lf), Ex[Pglr-1::magi-1::gfp]: 2.143±0.1 µm, n = 202, p = 0.349, two-tailed t-test) Furthermore, GLR-1::GFP clusters in Pglr-6::magi-1 animals reverted to nearly the size observed in unconditioned animals after 30 minutes of recovery, suggesting that expression of MAGI-1 in RIA alone was not sufficient to mediate a persisting size reduction of GLR-1 clusters at AVA and AVD synapses in the VNC . Finally, all aspects of the conditioned changes in GLR-1 clustering were rescued when magi-1 was simultaneously expressed in RIA, AVA and AVD using the glr-1 and glr-6 promoters . Thus, MAGI-1 plays distinct functions in RIA, AVA and AVD neurons during learning and memory. Expression of MAGI-1 in RIA induces during associative learning the remodeling of GLR-1 containing synapses in AVA and AVD in a cell non-autonomous fashion. At the same time, MAGI-1 acts cell-autonomously in AVA and AVD to consolidate the conditioned changes in GLR-1 clustering during memory formation.We next tested if MAGI-1 regulates GLR-1 distribution in the VNC of wild-type and itioning . We thenitioning . Expresslearning  but also mutants  (magi-1(romoters  (1.3±0.0in vitro nor in vivo (data not shown). Since the vertebrate MAGI protein interacts with β-catenin C. elegans MAGI-1 might bind to the β-catenin homolog HMP-2, the only one of the three C. elegans β-catenins that is predicted to contain a C-terminal PDZ binding motif . GST pull-down experiments revealed binding of HMP-2 to MAGI-1 (magi-1(lf) mutants (magi-1(lf) mutants  mutant worms. Furthermore, loss of MAGI-1 has additional effect on neuronal plasticity besides the demonstrated role in non-associative learning C. elegans and humans.In summary, we demonstrate that RIA, AVA, AVD and likely AVE interneurons are responsible for different aspects of associative learning in  elegans . While pC. elegansC. elegans Bristol strain, variety N2, was used as the wild-type reference strain in all experiments. Alleles and transgenes used were magi-1(tm446), nuIs25akEx211; lin-15(n765ts), magi-1(zh66), magi-1(zh66); nuIs25, magi-1(zh66); utrEx9, magi-1(zh66); utrEx2, magi-1(zh66); utrEx3, magi-1(zh66); utrEx12, magi-1(zh66); utrEx10, magi-1(zh66); utrEx13, magi-1(zh66); utrEx14, magi-1(zh66); nuIs25; utrEx4, magi-1(zh66); nuIs25; utrEx5, magi-1(zh66); nuIs25; utrEx6, magi-1(zh66); utrEx8, magi-1(zh66); utrEx17, magi-1(zh66); utrEx19 . Transgenic lines were generated by injecting the indicated DNA fragments at a concentration of 50-100 ng/µl into both arms of the syncytial gonad as described psur-5::mDsRed was used as a transformation marker at 10 ng/µl concentration.Standard methods were used for maintaining and manipulating magi-1, a library consisting of ∼5*105 mutagenized F1 animals was screened as described 2 UV, allowed to recover for 16 hours and five P0 animals were plated on each of 960 NGM plates. Cultures were grown and one third of the animals were harvested for DNA isolation. To identify the plate containing the magi-1(zh66) deletion, DNA extracts from 12 cultures was pooled and each pool was tested by nested PCR with outer primers OAS-364 [5′TTCCCGGGTGAAATTGCGACCCAACG TTG3′], OAS-422 [5′CAGGATGATGTCCTCATCCTATC3′], and inner primers OAS-423 [5′GCTCCGTCGACTAGTTCGAGTAC3′], OAS-424 [5′CCATCGGAAGAACTGGTCCAG CTG3′]. The remaining animals from the culture positive for the zh66 deletion were subjected to three rounds of sib-selection until a single homozygous magi-1(zh66) animal had been identified. Before further analysis, the magi-1(zh66) mutation was backcrossed five times against N2 animals.To isolate a deletion mutation in Olfactory conditioning was assessed as described 2PO4/K2HPO4 pH = 6.0, 1 mM CaCl2, 1 mM MgSO4, 2% agar). Naïve and conditioned worms were given a choice between a spot of 0.1% (vol/vol) DA or IA in ethanol with 20 mM sodium-azide and a counter spot with ethanol and sodium-azide. After one hour the animals were counted and a chemotaxis index was calculated as described Chemotaxis to water-soluble compounds was assessed as described with some modifications 2PO4/K2HPO4 pH 6.0, 1 mM CaCl2 and 1 mM MgSO4), either containing 25 mM NaCl or no salt. Adjacent quadrants were connected with a thin layer of molten agar. A population of well-fed, young adults was washed three times with CTX buffer  and 100−200 worms were placed at the intersection of the four quadrants. The distribution of the worms over the four quadrants was determined after 10 minutes. For NaCl conditioning, animals were exposed to 25 mM NaCl in CTX buffer for 4 hours.A steep thermal gradient on a thin agar plate was established as described previously Locomotory rate assays were performed on a bacterial lawn as described GFP (or GFP variants)-tagged proteins were detected with a Zeiss Axiovert 200M LSM 5 Pascal confocal microscope. Animals were immobilized with sodium-azide and GLR-1::GFP was recorded posterior to the vulva, along the z-axis. Quantification of GLR-1::GFP cluster size was performed on the projected z-sections using Openlab 5.0 software package (Improvision).C. elegans genomic DNA. Primer sequences are available on request. GST-fusion proteins were expressed in E. coli and purified on glutathione sepharose using standard protocols. After incubation with total worm protein extracts prepared by lysing worms in lysis buffer , bound MAGI-1::GFP proteins were detected on Western blots using anti-GFP antibodies (Roche).Promoter-gene fusions were generated by PCR fusion as described Figure S1(0.30 MB DOC)Click here for additional data file.Figure S2(0.13 MB DOC)Click here for additional data file.Figure S3(0.20 MB DOC)Click here for additional data file.


C. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation.mab-31, recovered from a genome-wide RNAi screen. mab-31 mutants showed ray cell cluster patterning defect and mis-specification of the ray identity. mab-31 encodes a nuclear protein expressed in descendants of ray precursor cells impacting on the ray cell's clustering properties, orientation of cell division plane, and fusion of structural cells. Genetic experiments also establish its relationship with other Sma/Mab pathway components and transcription factors acting upstream and downstream of the signaling event.We report here the characterization of a new member of the Sma/Mab pathway, mab-31 function is indispensable in Sma/Mab signal recipient cells during sensory rays specification. Both mab-31 and sma-6 are required in ray lineage at the late larval stages. They act upstream of C. elegans Pax-6 homolog and repress its function. These findings suggested mab-31 is a key factor that can integrate TFG-β signals in male sensory ray lineage to define organ identity. C. elegans Small and Drosophila mad) proteins . These phenotypes are all specific, as they resemble those displayed by mab-31(RNAi) animals, and all abnormal phenotypes could be rescued by the functional transgene, pmab-31(2 kb)::mab-31cDNA.Each wild-type ray consists of dendritic processes of two ultra-structurally distinct sensory neurons, RnA and RnB. The dendritic endings of these neurons are held at an opening to the environment by a support cell, i.e. the structural cell (Rnst) Figure . Structu, E1-GFP  and ppkd-2::gfp2 , which m) Figure  and mab-) Figure , the str6 Figure . In bothd Figure . These rhenotype . mab-31, and mab-31(tm2718) animals. To achieve this, we traced the Rn cells lineage with an apical junction marker ajm-1::GFP. We were particularly interested in differentiation of R6 and R7, since rays 6-7 fusion was the most obvious phenotype in both mab-31 and other sma mutants. When R(6/7).a and R(6/7).p cells were born, there was no detectable difference in wild-type  also, inferring that the mab-31 gene probably acts in the same canonical dbl/sma pathway.Ray identity determination is a dynamic process of ray precursor (Rn) cells differentiation in late larval stages of e Figure , sma-6  (65%) and sma-6(wk7) (45%) mutants (N = 50). For example, the ray 7 papillae in mutant animals were not found in their native position but often at an ectopic position just next to the ray 6 papillus. This displacement is also consistent with our observation that ray 6 and 7 cell groups were next to each other during ray development as described above  and sma-6(wk7) carry recessive null mutations for ray patterning defects. We compared the frequency of rays 6-7 fusion phenotype in each single mutant as well as in double mutants. Worms doubly mutant for mab-31 and sma-6 do not exhibit an enhanced frequency of fusion for rays 6-7 (58%) compared to mab-31 single mutants  showed both Sma and Mab phenotypes. However, in a weak loss-of-function allele sma-6(e1482), the male tail defect could not be observed. It was suggested that the residual activity of sma-6(e1482) is sufficient for the male tail development, but its level is below the thresholds required for body size regulation  JcIs1IV; him-5(e1490)V,KC8420: sma-6(wk7)II; him-5(e1490) wxIs52[pB0024.14b::gfp+ppkd-2::gfp2]V,KC8470: mab-31(tm2718) wxIs29(pRF4+pKS+T24C2sphIGFPNLS-)I;him-5(e1490)V,KC8480: mab-31(tm2718)I;him-5(e1490)V;wxIs52[pB0024.14b::gfp+ppkd-2::gfp2]V,KC8490: mab-31(tm2718)I; jcIs1IV; him-5(e1490)V,KC8090: him-5(e1490)V; wxEx64[pRF4+pmab-31::gfp(2.0 k)],KC9640: him-5(e1490)V; wxEx123[pRF4+pmab-31(2 kb)::cDNA::GFP], andKC9650: mab-31(tm2718)I; su93JcIs1IV;him-5(e1490)V; wxEx122[pcrm-1b::gfp+pmab-31(2 kb)::cDNA::GFP]E. coli HT115(DE3) carrying the empty RNAi vector L4440 was fed to the same strain as a negative control.RNAi clone I-7J10 was from the Ahringer RNAi library (MRC Geneservice) . The ideN > 100). Of the transgenic strains, only transformed worms carrying the markers were imaged and scored. For the body length measurement, L4 hermaphrodites grown at 20°C were transferred to fresh NGM plates. Five days later, 100 F1 adult hermaphrodites were randomly photographed under 5× or 10× objectives with SPOT RT camera (Diagnostic Instruments). Their body length was measured through a scale adjusted with SPOT RT v3.4.5 software and was presented as mean length with standard error.The male tails were examined by Nomarski microscopy (ajm-1::GFP reporter (Strain SU93) were observed and recorded over a period of 4-5 hours.Worms were mounted on 2% agarose pads in 2 μl of M9 mixed with a smear of OP50 bacteria. A 12 × 12 mm cover slip was gently lowered onto the worm and divisions of the ray precursor cells marked by the expression of mab-31 translational GFP fusion construct was made using pPD vectors kindly supplied by the Fire Lab (Stanford University) and verified by sequencing. GFP from pPD95.75 was excised using KpnI and inserted into the KpnI site at the 3' end of mab-31 cDNA amplified from wild-type animals, with stop codon mutated. This GFP-tagged 3' mab-31 cDNA was inserted into a vector carrying 2 kb of mab-31 upstream sequence, which is amplified with primers KC 1058 (5' GCTCGAATAACCTGG 3') and KC1059 (5' AGGTCCGCCCACTTGTT CC 3'), to generate a full-length mab-31 cDNA GFP-tagged rescuing construct, MAB-31A::GFP. The transcriptional reporter mab-31::gfp was generated by utilizing the same 5' upstream sequence and cloned into pPD95.75 vector. Germ line transformation with different constructs was carried out as described [rol-6 gene or crm-1b::gfp as the selection marker. In general, at least three transgenic lines were generated for each plasmid construct and rescue or expression data were collected and pooled for comparison.A escribed . All tramab-31(tm2718) was outcrossed with him-5(e1490) three times. Homozygosity of mab-31 mutation was confirmed by PCR using primer sets (KC1599 5' ATGAACTCGCTCGAGTACC 3' and KC1601 5' GGTACCTTCTGATAAAGCAA ACGG ATTA 3'). Double mutants containing mutations in sma-4, mab-18, mab-21, unc-130 and mab-31 were constructed by crossing mab-31(tm2718) heterozygote males with homozygous hermaphrodites of each mutant. For double mutants with either sma-4 or unc-130, Sma or Unc mutants were carefully counted and placed onto separate plates. Their mab-31 genotypes were confirmed by PCR. Sma or Unc hermaphrodites that bore a mab-31(tm2718) homozygous gene were selected as double mutants. For double mutants with either mab-18 or mab-21, F2 hermaphrodites were placed onto separate plates and their mab-31 genotypes were confirmed by PCR after F3 progeny were laid. The plates bearing males with Mab-21 or Mab-18 phenotype were confirmed as double homozygous mutants.YFW, QS, and JWC contributed equally in conducting most of the experiments in this study. JKC examined the cellular defect of Sma/Mab mutants and carried out lineage analysis. YFW and KLC supervised the project and prepared the manuscript. All authors have read and approved the final manuscript.mab-31 gene structure and protein sequencesSchematic representation of . mab-31 gene products are embedded in ~10.5 kb intron 9 of cogc-1. Mutation tm2718 deleted coding sequence of mab-31a, but it does not affect the transcript level of mab-31b, a transcript irrelevant to the ray fusion phenotype. B) Sequence alignment of nematodes MAB-31 proteins  was shown by ClustalX. The conserved nuclear localization signal (NLS) (PredictNLS online) and deleted region of MAB-31 protein in tm2718 mutant were predicted and labeled.Click here for file


Focal Adhesion Kinase (FAK) is recently reported to regulate insulin resistance by regulating glucose uptake in C2C12 skeletal muscle cells. However, the underlying mechanism for FAK-mediated glucose transporter-4 translocation (Glut-4), responsible for glucose uptake, remains unknown. Recently actin remodeling was reported to be essential for Glut-4 translocation. Therefore, we investigated whether FAK contributes to insulin-induced actin remodeling and harbor Glut-4 for glucose transport and whether downregulation of FAK affects the remodeling and causes insulin resistance.To address the issue we employed two approaches: gain of function by overexpressing FAK and loss of function by siRNA-mediated silencing of FAK. We observed that overexpression of FAK induces actin remodeling in skeletal muscle cells in presence of insulin. Concomitant to this Glut-4 molecules were also observed to be present in the vicinity of remodeled actin, as indicated by the colocalization studies. FAK-mediated actin remodeling resulted into subsequent glucose uptake via PI3K-dependent pathway. On the other hand FAK silencing reduced actin remodeling affecting Glut-4 translocation resulting into insulin resistance.The data confirms that FAK regulates glucose uptake through actin reorganization in skeletal muscle. FAK overexpression supports actin remodeling and subsequent glucose uptake in a PI3K dependent manner. Inhibition of FAK prevents insulin-stimulated remodeling of actin filaments resulting into decreased Glut-4 translocation and glucose uptake generating insulin resistance. To our knowledge this is the first study relating FAK, actin remodeling, Glut-4 translocation and glucose uptake and their interrelationship in generating insulin resistance. The uptake of glucose by skeletal muscle, a major insulin responsive organ, is mediated by the insulin-responsive glucose transporters, Glut-4 ,2. InsulIn order to better understand insulin resistance at a molecular level we have previously developed an insulin resistant skeletal muscle cell model by differentiating C2C12 skeletal muscle cells in chronic presence (MFI) and absence (MF) of insulin in serum free medium . The modFak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with focal adhesions and microtubules 2-deoxyglucose (2DOG), TEMED, anti-rabbit, anti-mouse IgG alkaline phosphatase and FITC conjugated were from Sigma Chemical Company . Anti-FAK antibody was from Santa Cruz Biotechnology . Bovine insulin, wartmanin and anti-Glut-4 antibody was purchased from Calbiochem . 4,6-diamidino-2-phenylindole (DAPI), Phalloidin Texas Red were purchased from Molecular Probes, Inc. TransFast transfection reagent was from Promega . RNAifect transfecton reagent was from Qiagen GmbH (Germany). All the other reagents unless attributed specifically were from Sigma Chemical Company.DMEM and trypsin-EDTA were purchased from Gibco BRL . FCS was from Biological Industries . Nutrient Mixture F-12 Ham, MCDB 201 medium, BSA (cell culture grade), 2-deoxyglucose. Glut-4: Glucose transporter-4. siRNA: small interfering RNA. WM: wartmanin.FAK: focal adhesion kinase. PI3K: phosphatidylinositol 3'-kinase. GFP: green fluorescent protein. DAPI: 4,6-diamidino-2-phenylindole. 2DOG: [The authors declare that they have no competing interests.BB designed and executed experiments, analysed data and drafted the manuscript. CSD conceived the study, designed, coordinated experiments, written and finalized the manuscript. All authors read and approved the final manuscript.C2C12 cells expressing GFP-Glut-4. C2C12 cells were transfected with GFP-Glut-4 plasmid and expression of Glut-4 (GFP: Green), Actin  and Nucleus (DAPI: Blue) were examined in proliferated and differentiated cells. No green fluorescence was observed in untransfected, proliferated C2C12 wild type cells (A) whereas transfected cells, expressing GFP-Glut-4 (C2Glut-4wt/+), emitted green fluorescence in (B) proliferated C2Glut-4wt/+ and (C) Differentiated C2Glut-4wt/+ cells. Intact nucleus as observed in DAPI stained transfected cells confirmed no cell death due to transfection. Images are representative of multiple fields of 3 independent experiments. Bar corresponds to 10 μm.Click here for filewt/+ cellsInsulin-mediated translocation of GFP-Glut-4 molecules in C2Glut-4. C2Glut-4wt/+ cells were differentiated and stimulated with and without insulin (100 nM) for 30 min and translocation of Glut-4 were examined. In absence of insulin Glut-4 molecules remained concentrated in cytoplasm near the nucleus (A) whereas they dispersed in the cytoplasm in the process of translocation to the membrane after insulin stimulation (B). Blue arrow head indicates Glut-4 molecules; Red arrow head indicates the peripheral region without Glut-4 molecules. Images are representative of multiple fields of 3 independent experiments. Bar is 5 μm, 10 μm for inset.Click here for filewt/+ cellsTransfection of FAK wild type plasmid and siRNA for FAK in C2Glut-4. C2Glut-4wt/+ cells were transfected with FAK wild type plasmid and FAK specific siRNA (400 nM). (A) Cells were differentiated, lysed and western immunoblotted with anti-FAK and anti-Glut-4 antibodies. Blots were stripped and re-probed with anti-actin antibody. Lane 1: C2C12 wild type myotubes, Lane 2: C2Glut-4wt/+ cells, Lane 3: C2Glut-4wt/+FAKwt/+ and Lane 4: C2Glut-4wt/+FAKwt/-. (B) Morphological examination of differentiated transfected and untransfected cells. No change in cellular morphology of transfected and untransfected cells was observed. Images are representative of multiple fileds of 3 experiments. Bar is 20 μm.Click here for filewt/+ cellsEffect of insulin on actin remodeling and its colocalization with Glut-4 in C2Glut-4. C2Glut-4wt/+ cells were differentiated under MF and MFI condition, serum starved (4 h) and stimulated with and without 100 nM insulin for 30 min at 37°C followed by fixation and permeabilization. Actin filaments, nucleus, Glut-4 were labeled with Phalloidin Texas Red, DAPI and GFP respectively. Images were captured from different fields and a representative image of 3 experiments is presented. Bar is 5 μm. (A) C2Glut-4wt/+ cells differentiated under MF condition  C2Glut-4wt/+ cells differentiated under MF condition stimulated with insulin  C2Glut-4wt/+ cells differentiated under MFI condition  C2Glut-4wt/+ cells differentiated under MFI condition stimulated with insulin  were differentiated under MF and MFI condition, serum starved (4 h) and stimulated with and without 100 nM insulin for 30 min at 37°C followed by fixation and permeabilization. Actin filaments, nucleus, Glut-4 were labeled with Phalloidin Texas Red, DAPI and GFP, respectively. Images were captured from different fields and a representative image of 3 experiments is presented. Bar is 5 μm. (A) C2Glut-4wt/+FAKwt/+ cells differentiated under MF condition  C2Glut-4wt/+FAKwt/+ cells differentiated under MF condition stimulated with insulin  C2Glut-4wt/+FAKwt/+ differentiated under MFI condition  C2Glut-4wt/+FAKwt/+ cells differentiated under MFI condition stimulated with insulin  were differentiated under MF and MFI condition, serum starved (4 h) and stimulated with and without 100 nM insulin for 30 min at 37°C followed by fixation and permeabilization. Actin filaments, nucleus, Glut-4 were labeled with Phalloidin Texas Red, DAPI and GFP, respectively. Images were captured from different fields and a representative image of 3 experiments was presented. Bar is 5 μm. (A) C2Glut-4wt/+FAKwt/- cells differentiated under MF condition  C2Glut-4wt/+FAKwt/- cells differentiated under MF condition stimulated with insulin  C2Glut-4wt/+FAKwt/- differentiated under MFI condition  C2Glut-4wt/+FAKwt/- cells differentiated under MFI condition stimulated with insulin (represent Fig. Click here for filePossible signal pathway for FAK-regulated insulin signaling.Click here for file


The EVI-1 gene was originally detected as an ectopic viral insertion site and encodes a nuclear zinc finger DNA-binding protein. Previous studies showed restricted EVI-1 RNA or protein expression during ontogeny; in a kidney and an endometrial carcinoma cell line; and in normal murine oocytes and kidney cells. EVI-1 expression was also detected in a subset of acute myeloid leukaemias (AMLs) and myelodysplasia. Because EVI-1 is expressed in the urogenital tract during development, we examined ovarian cancers and normal ovaries for EVI-1 RNA expression using reverse transcription polymerase chain reaction (RT-PCR) and RNAase protection. Chromosome abnormalities were examined using karyotypes and whole chromosome 3 and 3q26 fluorescence in situ hybridisation (FISH). RNA from six primary ovarian tumours, five normal ovaries and 47 tumour cell lines  was studied. Five of six primary ovarian tumours, three of five normal ovaries and 22 of 25 ovarian cell lines expressed EVI-1 RNA. A variety of other non-haematological cancers also expressed EVI-1 RNA. Immunostaining of ovarian cancer cell lines revealed nuclear EVI-1 protein. In contrast, normal ovary stained primarily within oocytes and faintly in stroma. Primary ovarian tumours showed nuclear and intense, diffuse cytoplasmic staining. Quantitation of EVI-1 RNA, performed using RNAase protection, showed ovarian carcinoma cells expressed 0 to 40 times the EVI-1 RNA in normal ovary, and 0-6 times the levels in leukaemia cell lines. Southern analyses of ovarian carcinoma cell lines showed no amplification or rearrangements involving EVI-1. In some acute leukaemias, activation of EVI-1 transcription is associated with translocations involving 3q26, the site of the EVI-1 gene. Ovarian carcinoma karyotypes showed one line with quadruplication 3(q24q27), but no other clonal structural rearrangements involving 3q26. However, whole chromsome 3 and 3q26 FISH performed on lines with high EVI-1 expression showed translocations involving chromosome 3q26. EVI-1 is overexpressed in ovarian cancer compared with normal ovaries, suggesting a role for EVI-1 in solid tumour carcinogenesis or progression. Mechanisms underlying EVI-1 overexpression remain unclear, but may include rearrangements involving chromosome 3q26.


The result suggests a weakintramolecular metal-metal interaction in dppm(AuSR∗)2 that correlates well with rate accelerationoccurring in the reaction of dppm(AuSR∗)2 with organic disulfides. Quantum yields for cis-dppee(AuX)2 complexes, (disappearance) Φ                , are significantlyhigher in complexes with a softer X ligand, a trend that correlates well with aurophilicity. This result alsosuggests that electronic perturbation caused by Au(I)-Au(I) interactions is important in explaining thereactivity of some dinuclear gold(I) complexes. The crystal structure for cis-dppee(Aul)2 shows shortintramolecular Au(I)-Au(I) interactions of 2.9526 (6) A°, while the structure of trans-dppee(AuI)2, shows intermolecular Au(I)-Au(I) interactions of 3.2292 (9) A°. The substitution of .As for P results in a ligand, cis-diphenylarsinoethylene(cis-dpaee), that is photochemically active, in contrast to the cis-dppee ligand.The complexes, cis-dpaee(AuX)2, are also photochemically active but with lower quantum yields than thecis-dppee(AuX)2 complexes.Cyclic voltammetry (CV) experiments on LL(AuSR∗)


Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs  are characterized during the processes of cell spreading and membrane ruffling.We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling.Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes. Cell adhesion to an extracellular matrix (ECM) is a fundamental feature of multicellular organisms and is a critical component of development, tissue function and homeostasis. Adhesive interactions between cells and ECM are complex, requiring the regulation of integrin localization and activity, reorganization of the actin cytoskeleton and localized membrane remodeling. After engagement of an ECM substrate, integrin-mediated biochemical signaling induces the actin-driven formation of Rac1-dependent lamellipodia and/or Cdc42-dependent filipodia  fusion protein; SNARE: soluble NSF attachment protein receptor; TeTx-LC: tetanus toxin light chain; TGN: trans-Golgi network; VAMP: vesicle associate membrane protein.CHO: Chinese hamster ovary; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FBS: fetal bovine serum; FN: fibronectin; GFP: green fluorescent protein; NSF: MS conceived and carried out the immunoprecipitation, lammellipodia extension and immunofluorescence trafficking assays, and drafted the initial manuscript. QY conceived and performed the ruffling assays. MJK carried out the migration assays and contributed to the ruffling assays. DWM assisted in the immunoprecipitation assays. KCW contributed experimentally to the shRNA knock-down studies. AB generated several of the SNARE constructs and contributed to the spreading assays. MGC conceived/designed the study, analyzed/processed data, assembled figures and edited the manuscript. All authors read and approved the final manuscript.Quantification of SNAREs. (A and B) CHO-K1 cells were held in suspension after pretreatment with 1 mM NEM and 2 mM DTT (Susp), held in suspension after pretreatment with 1 mM NEM (Susp+NEM) or plated on FN (FN) for 20 min. (A) Graph shows the intensities of syntaxin13 bands from SNAP23 immunoprecipitates, normalized to samples adherent to FN (mean +/- SD of at least three experiments) (B) Intensities of syntaxin13 bands from VAMP4 immunoprecipitates, normalized to samples adherent to FN (mean +/- SD of at least three experiments). (C and D) CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, lysed and VAMP4 (C) or VAMP3 (D) were immunoprecipitated. Graphs show intensities of SNAP23 bands from VAMP4 immunoprecipitates (C) or VAMP3 immunoprecipitates (D), normalized to non-induced samples. Data represent means +/-SD from at least 3 independent experiments.Click here for fileComplex containing Syntaxin13 and SNAP23 does not form after lysis of cells. Cells transfected with FLAG-SNAP23 or GFP-syntaxin13 alone were held in suspension and lysed. After lysis, the FLAG-SNAP23-containing and GFP-syntaxin13-containing lysates were mixed for 1 h (In Vitro Mix) followed by immunoprecipitation with anti-GFP antibody overnight. Cotransfected cells were held in suspension (Susp) or plated on FN (FN), lysed and then immunoprecipitations were done with anti-GFP antibody. GFP immunoprecipitates were probed for the presence of FLAG-tagged SNAP23, upper blot, using anti-FLAG antibody, and the same membrane was reprobed for GFP-syntaxin13, lower blot, using anti-GFP antibody. "Bead+Lys" is eluate from sepharose beads incubated with lysate and "GFP Ab" is eluate from sepharose-coupled anti-GFP in lysis buffer.Click here for fileDominant-negative SNAREs bind cognate SNAREs and impair endogenous binding. CHO-K1 cells were transiently transfected with truncated GFP-tagged constructs of syntaxin13 (syn13cyto-GFP), VAMP3 (VAMP3cyto-GFP), VAMP4 (VAMP4cyto-GFP) or SNAP23 (SNAP23CΔ9-GFP), lysed and cognate SNAREs were immunoprecipitated with antibodies coupled to sepharose beads. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for inhibitor and endogenous SNARE binding. (A) Syntaxin13 western blot of SNAP23 immunoprecipitaes from syn13cyto transfected cells. (B) VAMP3 western blot of SNAP23 and syntaxin13 immunoprecipitaes from VAMP3cyto transfected cells. (C) VAMP4 western blot of SNAP23 and syntaxin13 immunoprecipitaes from VAMP4cyto transfected cells. (D) SNAP23 western blot of VAMP3 and syntaxin13 immunoprecipitaes from SNAP23CΔ9 transfected cells.Click here for fileIn cells expressing dominant-negative SNARE domains integrin co-localizes with Rab4. CHO-K1 cells were transiently transfected for 22 h with GFP [A-D] or truncated GFP-tagged constructs of VAMP3 (VAMP3cyto) [E-H], syntaxin13 (syntaxin13cyto) [I-L], VAMP4 (VAMP4cyto) [Q-T] or 15 h with SNAP23 (SNAP23CΔ9)[M-P]. Cells were labeled with anti-β1 integrin antibody in serum free media for 1 h to allow internalization of the label. Cells were then washed in 0.2 M glycine, pH 2.5, fixed and stained for the labeled integrin  and Rab4 . Overlays of the Rab4 and β1 integrin staining are shown in D, H, L, P and T. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.Click here for fileDominant-negative SNARE domains inhibit trafficking of transferrin into a perinuclear compartment. CHO-K1 cells were transiently transfected for 22 h with truncated GFP-tagged constructs of VAMP3 (VAMP3-cyto) [A], syntaxin13 (syntaxin13-cyto) [C], VAMP4 (VAMP4-cyto) [G], or 15 h with SNAP23 (SNAP23CΔ9) [E]. To load the perinuclear recycling compartment with Tfn, cells were serum starved for 1 hr followed by incubation with 30 μg/mL rhodamine-Tfn in serum-free media for 15 min. Coverslips were briefly washed with 0.2 M glycine, pH 2.5, to remove surface bound Tfn, washed three times with PBS and fixed in 4% paraformaldehyde in PBS. Arrows in B, D and F point to perinuclear, rhodamine-Tfn-containing compartment in non-transfected control cells. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μmClick here for file


Background: The cytotoxic T lymphocyteassociatedantigen 4 gene (CTLA-4) encode theT cell receptor involved in the control of T cellproliferation and mediates T cell apoptosis.The receptor protein is a specific T lymphocytesurface antigen that is detected on cells onlyafter antigen presentation. Thus, CTLA-4 isdirectly involved in both immune and autoimmuneresponses and may be involved in thepathogenesis of multiple T cell-mediatedautoimmune disorders. There is polymorphismat position 49 in exon 1 of the CTLA-4 gene,providing an A-G exchange. Moreover, weassessed the CTLA-4 49 (Thr/Ala) polymorphismin diabetic patients and first-degree relativesas compared to control subjects.                    Research design and methods: Three loci were analysed in 62 type 1 diabetic patients, 72 firstdegreerelatives and 84 nondiabetic controlsubjects by means of PCR-RFLP.                    Results: A significant enrichment in DQB1alleles encoding for an amino acid differentfrom Asp in position 57 (NA) and DQA1 allelesencoding for Arg in position 52 wasobserved in diabetic subjects and first-degreerelatives as compared to controls. The genotypeand allele frequencies of these polymorphismsin type 1 diabetic patients and firstdegreerelatives differed significantly fromthose of controls (p< 0.001 and 0.05 respectively).CTLA-49 Ala alleles frequencies were75.8% in type 1 diabetic patients and 68.1% infirst-degree relatives in comparison to 35.7% incontrol subjects. The Ala/Ala genotype conferreda relative risk of 18.8 (p < 0.001).                    Conclusion: The CTLA-4 49 Ala allele confersan increased risk of type 1 diabetes, independentof age and HLA-DQ genetic markers.


The FONICAP group is screening, with randomised phase II studies, the activity of new chemotherapy programmes for advanced non-small-cell lung cancer (NSCLC) looking for regimens with > 30% activity. In the present study, three regimens were tested: MIP ; MIP-IFN ; and PC (cisplatinum 60 mg m-2 and carboplatin 400 mg m-2 on day 1 every 28 days). Overall 93 chemotherapy-naive patients were enrolled: 23 received MIP, 27 received MIP-IFN and 43 received PC. Eighty per cent of the patients had stage IV and 20% stage IIIb disease . Response rates were as follows: MIP = 9% (95% CI 1-28%), MIP-IFN = 7% (95% CI 1-24%) and PC = 14% (95% CI 5-28%). The overall median survival was 183 days. Grade III-IV leucopenia was observed in 36% of patients treated with MIP-IFN vs 10% in the other two arms, and thrombocytopenia grade III-IV was reported in nearly 10% of patients overall. In conclusion, (1) all three regimens investigated have poor activity (< 30%); (2) when tested in multicentre randomised phase II trials, MIP displays lower activity than in phase II trials; (3) PC has similar activity to other platinum-containing regimens; (4) randomised phase II studies are a reliable and quick method of determining the anti-tumour activity of novel chemotherapeutic regimens in NSCLC.


Interleukin-5 (IL-5) has been shown to be a selective eosinophilgrowth and differentiation factor. In the present study, the effectof recombinant human IL-5 on human eosinophil sulfidopeptideleukotriene production was investigated. IL-5 did not affectleukotriene synthesis in unstimulated eosinophils. However, IL-5potentiated leukotriene synthesis by eosinophils stimulated withserum treated zymosan (STZ) or the calcium ionophore A23187 by69% and 135%, respectively. The priming effect of IL-5was dose dependent, with significant stimulation occurring at 1 000U/ml for STZ and 100-1 000 U/ml for A23187. Pre-incubation with IL-5did not increase leukotriene synthesis further.