This BDNF-evoked IEG response required the transcription factor SRF (serum response factor). Notably, ephrin-A5 suppressed a BDNF-evoked neuronal IEG response, suggesting a role of Eph receptors in modulating gene expression. In opposite to IEGs, long-term ephrin-A5 application induced cytoskeletal gene expression of tropomyosin and actinin. To uncover specific Eph receptors mediating ephrin-As impact on neurotrophin signaling, EphA7 deficient mice were analyzed. In EphA7 deficient neurons alterations in growth cone morphology were observed. However, ephrin-A5 still counteracted neurotrophin signaling suggesting that EphA7 is not required for ephrin and BDNF crosstalk. In sum, our data suggest an interaction of ephrin-As and neurotrophin signaling pathways converging at ERK signaling and nuclear gene activity. As ephrins are involved in development and function of many organs, such modulation of receptor tyrosine kinase signaling and gene expression by Ephs might not be limited to the nervous system.During brain development, growth cones respond to attractive and repulsive axon guidance cues. How growth cones integrate guidance instructions is poorly understood. Here, we demonstrate a link between BDNF (brain derived neurotrophic factor), promoting axonal branching and ephrin-A5, mediating axonal repulsion via Eph receptor tyrosine kinase activation. BDNF enhanced growth cone filopodial dynamics and neurite branching of primary neurons. We show that ephrin-A5 antagonized this BDNF-evoked neuronal motility. BDNF increased ERK phosphorylation (P-ERK) and nuclear ERK entry. Ephrin-A5 suppressed BDNF-induced ERK activity and might sequester P-ERK in the cytoplasm. Neurotrophins are well established stimulators of a neuronal immediate early gene (IEG) response. This is confirmed in this study by e.g.  During brain development, axons encounter attractive and repulsive guidance cues, whose interplay instructs growth cones with directional information, thereby ensuring target recognition. For instance, axons initially overshoot their final termination zone until, later on, such ectopic arborizations are eliminated and only branches in the prospective termination zone are stabilized, a process termed axon pruning Here, we studied axon guidance responses elicited by ephrin-A and BDNF co-stimulation of mouse primary neurons. Eph family members signal bi-directionally. In EphA forward signaling, ephrin-A ligands can activate multiple Eph receptor tyrosine kinase receptors  in a highly promiscuous manner. This usually results in contact-mediated repulsion, e.g. growth cone collapse Egr1, c-fos, Arc) and actin cytoskeletal gene responses Srf mutants, cell migration BDNF is considered an attractive axon guidance cue, e.g. promoting retinal axon branching So far, an interaction of EphA forward signaling and neurotrophins has not been analyzed in detail. In contrast, a crosstalk between EphA reverse and neurotrophin signaling is well documented in vitro. Ephrin-A5 application attenuated BDNF-stimulated ERK activity by suppression of ERK phosphorylation. Ephrin-A5 incubation resulted in co-localization of phosphorylated ERK and Eph receptors in the neurites. Eph forward signaling suppressed a BDNF-evoked IEG response conveyed by SRF. As demonstrated by EphA7 deficient neurons, EphA7 is dispensable for ephrin-A's influence on neurotrophin signaling.Here, we demonstrate an interaction of ephrin-A and neurotrophin signaling in primary hippocampal and cortical neurons. Activation of Eph forward signaling by ephrin-A5 antagonized BDNF-enhanced neuronal motility Taken together, our data suggest a counteraction of neurotrophin signaling by ephrin-As. This finding might also be applicable to signaling processes regulated by Eph family members outside of the nervous system, e.g. in organogenesis and tumorigenesis.Srf (flex1neo/flex1neo) and Camk2α-iCre mice were bred to obtain Srf mutants  or control littermates  were cultured on poly-L-lysine  and laminin  coated coverslips (13 mm). Ephrin-A5-Fc (R&D systems) or Fc alone (Sigma) was applied to the culture medium at 1 µg/ml, both pre-clustered with 10 µg/ml anti-human IgG Fc-specific (Sigma) for 30 mins at 37°C. BDNF was applied at 10 ng/ml (6 cells/35 mm dish coated with poly-L-lysine at 10 µg/ml) were cultured for 3 div (days in vitro) prior to stimulation (see below). We used nucleofection  to deliver a constitutively-active mutant of MEK1 P1 hippocampal or E17.5 cortical cultures were incubated in NMEM/B27 medium as described previously 10 ng/ml . Stimula10 ng/ml . For exp10 ng/ml  and qRT-Cortical cultures were stimulated with 1 µg/ml clustered (see above) ephrin-A5-Fc and/or BDNF (10 ng/ml) in NMEM/B27 medium for the indicated time periods. Phosphatase inhibitors were applied for 15 mins prior to ephrin-A5-Fc and/or BDNF stimulation: sodium vanadate , okadaic acid , PhosStop . Cells were lysed in 100 mM Tris pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% SDS and protease inhibitors (Roche). Samples were resolved on 10-12% SDS-PAGE, followed by transfer on PVDF membranes (Amersham). After blocking, first antibodies were applied overnight at 4°C: rabbit anti-ERK , rabbit anti-P-ERK , mouse anti-GAPDH (1∶50000), rabbit anti-TrkB , rabbit anti-P-TrkB , mouse anti-EphA4 , P-EphA3 . Detection of first antibodies involved horseradish-peroxidase conjugated secondary antibodies (1∶5000) and the ECL Western Blotting Substrate (Pierce).Gapdh RNA levels. Primer sequences can be obtained upon request.Cultures were stimulated with 2.5 ng/ml BDNF and/or 1 µg/ml pre-clustered ephrin-A5-Fc (see above). Total RNA was isolated with the RNeasy kit (Qiagen). Reverse transcription was performed with 1 µg RNA using reverse transcriptase (Promega) and random hexamers. qRT-PCR was performed on ABI PRISM 7700 Sequence Detector with the Power PCR SYBR green PCR master mix (Applied Biosystems). Expression was determined in relation to Cells were fixed for 15 minutes in 4% PFA/5% Sucrose/PBS, permeabilized for 5 minutes in 0.1% Triton-X-100/PBS and blocked for 30 minutes in 2% BSA/PBS. Primary antibodies were incubated overnight at 4°C as follows: mouse α-ß-tubulin , mouse α-class III ß-tubulin , rabbit anti-ERK , rabbit anti-P-ERK . First antibodies were detected with Alexa 488 or 546-conjugated secondary antibodies . Cells were stained for F-actin with Texas Red-X Phalloidin .For visualization of P-ERK and Eph receptors, cultures were incubated with 1 µg/ml pre-clustered ephrin-A5 for 20 minutes, followed by fixation with 4%PFA/sucrose for 15 minutes. Ephrin-A5 bound to Eph receptors was visualized with anti-Fc Cy3 conjugated antibodies (1∶300 in block). Subsequently, P-ERK staining was performed as indicated above.Pictures were acquired on a Zeiss Axiovert 200M or Zeiss LSM confocal microscope using an Axiocam camera and Axiovision software. We used 10x, 20x and 63x Zeiss objective lenses with numerical apertures of 0.3, 0.8 and 1.3 (oil), respectively. Pictures were further processed using Photoshop software (Adobe).For data in P≤0.05, 0.01, 0.001. Error bars indicate standard deviation (s.d.) if not indicated otherwise.Statistical significance was assessed by using a two-tailed t-test (Excel software) with *, **, *** representing To study an interplay between ephrin-A and BDNF stimulated neuronal signaling, we investigated growth cone morphology  and neurSrf mutant growth cones lacked filopodia, therefore appearing in a “rounded” shape . In agreement with our data, EphA activation also decreased ERK signaling in hippocampal neurons Application of BDNF alone expectedly increased P-ERK levels . NotablyOne way by which ephrin-A5 might inhibit ERK phosphorylation is recruitment of phosphatase activity, which has been implicated as downstream effector of an EphA mediated growth cone collapse Subsequently to ERK phosphorylation, neurotrophins stimulate cytoplasmic to nuclear P-ERK translocation allowing for modulation of neuronal gene expression In sum, Eph forward signaling impaired BDNF mediated P-ERK activation.Srf mutant embryos were treated for 20 minutes or 4h with ephrin-A5, BDNF or both substances applied simultaneously. Subsequently, IEG mRNA levels were assessed by quantitative real-time PCR.Nuclear P-ERK enhances gene transcription by activating many transcription factors, including SRF c-fos  of Semaphorin 3A, an established growth cone collapsing agent Srf mutant neurons, suggest that the BDNF induced IEG response requires SRF. This is supported by a reduced induction of the IEGs Egr1, Egr2, Arc , the F-actin cross-linker alpha-actinin 1 (Actn1) and the motor protein dynein light chain 1  were analyzed. mRNA levels of other cytoskeletal genes were not altered by any of the guidance cues . Short-term stimulation with guidance cues did not result in major alterations of cytoskeletal mRNA levels except for upregulation of Tpm1 by ephrin-A5  transducing ephrin-A5's impact on neurotrophin signaling, EphA7 was further analyzed . EphA7 iEpha7 mutant neurons . In contrast, growth cone morphology of s arrows  was altes arrows . EphA7 ds arrows . Althougs arrows , filopods arrows .Epha7 mutant neurons towards ephrin-A5 was tested in the growth cone collapse assay  and individual receptor activation was not impaired by BDNF and ephrin-A5 co-application . This arc-fos and Egr1 can stimulate neuronal motility c-fos and Egr-1 emerges Arc as an interesting IEG relevant to neuronal motility and synapse formation Signaling by many receptor tyrosine kinases modulates gene activity. Contrastingly, Eph receptor signaling has so far not been unambiguously associated with gene transcription in neurons. We demonstrated that ephrin-A5 can suppress BDNF-mediated gene expression . This, aInduction of all IEGs required the transcription factor SRF . So far,Eph activation does not only counteract neurotrophin signaling as shown in this study and by others Fig. S1BDNF stimulated neurite branching is antagonized by Eph forward signaling. Cultures were incubated with ephrin-A5 and BDNF at the indicated combinations for three days, followed by staining for F-actin (red) and microtubules (green).  Untreated wild-type neurons (A) protruded multiple neurites and neurite length was increased compared to SRF-deficient neurons (B).  BDNF alone enhanced neurite numbers in wild-type (arrows in C), but not Srf mutant neurons (D).  Ephrin-A5 did not alter neurite numbers in wild-type (E) or SRF-deficient (F) neurons.  Ephrin-A5 antagonized the BDNF-stimulated increase in neurite branching in wild-type neurons (G). In Srf mutant neurons, no modulation of neurite numbers by ephrin-A5 and BDNF co-application was observable (H).  Quantification of average neurite (I) and branch (J) number. Statistical significance was calculated in relation to wild-type/control treatment. (K) BDNF-mediated increase in neurite number requires MEK as revealed by U-0126 application, a MEK inhibitor. *, P<0.05; **, P<0.01; ***, P<0.001. Error bars represent s.d. Scale-bar (A-H)  =  50 µm.(DOC)Click here for additional data file.Fig. S2Unlike ephrin-As, the repulsive guidance cue semaphorin-3A does not antagonize BDNF-meditated IEG induction. Cortical neurons were stimulated for 20 mins with 2.5 ng/ml BDNF, supernatant of HEK293 cells transfected with either GFP  or semaphorin 3A (sema3A) or BDNF together with sema3A. Subsequently, RNA was isolated and cDNA was subjected to qPCR using primers to IEGs indicated. Growth cone collapse experiments were performed in parallel to confirm repulsive activity of sema3A supernatants used in qPCR experiments. Numbers in bars indicate numbers of independent cultures used. The IEGs Arc (A), c-fos (B), Egr1 (C) and Egr2 (D) were induced by BDNF. Interestingly, Arc (A) and Egr2 (D) were slightly induced by semaphorin 3A alone. Co-application of BDNF and sema3a resulted in robust induction of all IEGs tested to an extent comparable to BDNF alone. Thus, the repulsive guidance cue semaphorin 3A does not, unlike ephrin-A5, suppress BDNF-induced IEG mRNA levels.(DOC)Click here for additional data file.Fig. S3Constitutively-active MEK1 (caMEK1) elevates P-ERK levels. Neurons were electroporated with indicated amounts of a plasmid expressing caMEK-FLAG. After two days in culture, cell lysates were prepared and subjected to Western Blotting with antibodies indicated. P-Erk levels were increased by overexpression of caMEK compared to mock-electroporated neurons.(DOC)Click here for additional data file.Fig. S4Alterations in cytoskeletal gene expression upon exposure to ephrins and BDNF. Wild-type cortical cultures were treated for 20 minutes  or 16h  with guidance cues as depicted, followed by quantification of mRNA levels of indicated genes by qRT-PCR.  Tropomyosin1 (Tpm1) was up-regulated within 20 minutes by ephrin-A5 alone (A). 16h exposure to BDNF, ephrin-A5 alone and both together resulted in elevated Tpm1 mRNA levels (B).  Tropomyosin2 (Tpm2) was, within this 20 minute stimulation, not obviously altered by any treatment (C). Contrastingly, after 16h of incubation, BDNF slightly and ephrin-A5 alone or both together increased more robustly Tpm2 mRNA amounts (D).  Actinin1 (Actn1) was slightly, yet significantly, induced in wild-type neurons by ephrin-A5 and BDNF co-application, but none of the other treatments. With longer exposure, all three combinations of guidance cues elevated Actn1 mRNA levels (F).  Within 20 minutes of incubation, dynein light chain  was not changed by any guidance cue (G). After 16 h of application, BDNF slightly and ephrin-A5 and both together more pronounced, elevated Dnal1 mRNA abundance. *, P<0.05; **, P<0.01; ***, P<0.001. Error bars represent s.d.(DOC)Click here for additional data file.


BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a  BCR-ABL fusion gene. One-third of adult ALL patients with the Ph' chromosome show major (M)-BCR rearrangements (resulting in a 210-kDa protein), whereas two-thirds have minor (m)-BCR rearrangements (resulting in a 190-kDa protein). These chimeric proteins are hyperactive tyrosine kinases (TK) that are located in the cytoplasm, where they recruit downstream effectors of cell proliferation and survival. Chromosomal rearrangements other than t are found in ALL, although they are less frequent (http://AtlasGeneticsOncology.org).Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by the clonal expansion of lymphoid progenitors. Outcome is poorer in adults than in children, probably due to the higher frequency of Philadelphia (Ph') chromosome–positive ALL PCM1-JAK2 gene fusion resulting from t in eosinophilia-associated atypical CML, ALL, acute myeloid leukemia (AML), and T cell lymphoma BCR-JAK2 gene fusion as the result of t was found in atypical CML SSBP2-JAK2 in pre-B ALL PAX5- and STRN3-JAK2 in childhood ALL SEC31A-JAK2 in classical non Hodgkin lymphoma MLL, TCF3, TEL and BCR-ABL rearrangements JAK2 is a non-receptor TK protein that is essential for signaling through a variety of cytokine receptors and is required for normal hematopoiesis Patient written informed consent was obtained before bone marrow biopsies were taken. This protocol was carried out according to current Spanish legislation on clinical research in humans and was approved by the Hospital Universitario de la Princesa Clinical Investigation Ethics Committee .All experimentation with mice was carried out in accordance with institutional guidelines from the animal care and use committee from Centro de Investigaciones Tecnológicas y Medioambientales (CIEMAT) and approved by them with approval ID: HEM 4-09.3/mm3. The white blood cell count was 10.8×103/mm3 with 38% lymphocytes, 6% monocytes, 4% myelocytes and metamyelocytes, and 58% small lymphoblasts. Immunophenotyping of bone marrow disclosed B-lineage lymphoblasts . Karyotyping performed on the marrow aspirate revealed 49,XY,+X,+2,+4,−9,−11,+19,add(19)(q13),+20,−22,+mar in 24 of 25 metaphases examined. BCR-ABL and MLL rearrangements were not detected by standard fluorescence in situ hybridization (FISH). While reverse transcriptase polymerase chain reaction (RT-PCR) analyses for BCR-ABL rearrangements were negative, we found a new BCR-JAK2 transcript. The patient was diagnosed with ALL and started a standard high-risk ALL protocol. Hematologic, cytogenetic, and complete molecular remission (CMR) was achieved after five weeks, and the treatment proceeded through four standard consolidation cycles followed by an autologous peripheral blood stem cell transplant (SCT) with cyclophosphamide and total body irradiation (12 Gy). Low-dose interferon alpha was administered for five months as maintenance antileukemia therapy. More than six years later, the patient remains in complete remission.A 58-year-old male presented with asthenia, abdominal pain and slight hepatosplenomegaly. The blood count was abnormal with anemia of Hb 10.9 g/dl and a platelet count of 41×10BCR-JAK2 qualitative PCR  with BCR-Fw-T and JAK2-Rv-T primers. The PCR product was cloned in PCR2.1-TOPO  and sequenced using the d-rhodamine terminator cycle sequencing kit and an ABI PRISM 337 DNA sequencer (Applied-Biosystems). Sequences were aligned using BLAST and ClustalW applications (http://searchlauncher.bcm.tmc.edu/). BCR-JAK2 was quantified using fluorescence resonance energy transfer (FRET)-hybridization probes designed by TibMolbiol  and hybridized to a BCR sequence upstream of the BCR-JAK2 breakpoint by means of BCR-B and JAK2-3 primers. The PCR master mix was prepared with 0.3 µM of each primer and probe using LightCycler FastStart DNA MasterPLUS HybProbe (Roche Diagnostics), and the reaction was performed in duplicate on a LightCycler 2.0 (Roche Diagnostics). Data were normalized using BCR levels in the same samples. Quantitative PCR (qPCR) for Bcl-xL, Osm was performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) and Socs2 (NM_007706) expression was analysed using RT2 qPCR primer assay  following the manufacturer's protocols. Data were normalized using H3 (patient sample) or Gapdh (Ba/F3 cells) levels.Total RNA was isolated using Ultraspec  and reverse transcribed using the Gene-Amp Gold RNA PCR Core Kit . PCR for BCR-ABL p190 was carried out according to tive PCR . Long-raBCR  and bacterial artificial chromosome (BAC) clone flanking probes covering the whole JAK2 gene  version hg18 (March 2006). BAC clones were obtained from the BACPAC Resource Center at the Children's Hospital  and from the Sanger Institute Mapping Core Group at The Wellcome Trust Sanger Institute  . BAC DNA was isolated from 5-ml cultures using a standard miniprep procedure  and labelled by nick translation with SpectrumGreen-dUTP and SpectrumOrange-dUTP (Vysis). FISH was performed according to standard procedures. In all experiments, 100–150 interphase nuclei were scored, and no metaphases were observed.FISH was performed on bone marrow interphase nuclei using a probe set for ATCC) were cultured in DMEM  containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin and 4 mM L-glutamine at 37°C and 5% CO2. Murine Ba/F3 pro-B cells (ATCC) were grown in DMEM with 10 ng/ml of recombinant IL-3 . Cytokine-independent Ba/F3 cells transduced with TEL-JAK2 were a gift from Dr. G. Reuther . JAK2 inhibitor TG101209 was provided by Dr. Fuller . Stock solutions were prepared in dimethylsulfoxide (DMSO).HEK293T cells (BCR-JAK2 cDNA was subcloned into the expression vector pcDNA3.1 (Invitrogen) through an EcoR I site. For retroviral infections in Ba/F3 cells, cDNA was cloned into the EcoR I site of the pLZR CMV-IRES-EGFP (pLZR) vector  to obtain the pLZR-BJ-IRES-EGFP (pLZR-BJ) vector. The pLZR vector was used as a control. All constructs were verified by DNA sequencing.For expression in HEK293T cells, BCR-JAK2. At 24 and 48 h post-transfection, viral supernatants were harvested, filtered, and stored at −80°C. Ba/F3 cells were infected with retroviral supernatants preloaded four times during 30 min in Retronectin -coated plates. After 7 days, EGFP+ cells were sorted by flow cytometry using a FACSAria cytometer . For proliferative assays, 3×105 cells of Ba/F3-mock or Ba/F3-BJ cells were cultured with or without IL-3 (10 ng/ml) on 24-well plates and counted daily by trypan blue exclusion. TG101209 dose-dependent kinetic assays were performed as described 4 cells/ml for in vitro expansion. Cells were counted by trypan blue exclusion every week during 30 days.Transient transfections in HEK293T cells were performed with 3 µg of plasmid in Opti-MEM  using Lipofectin (Invitrogen) according to the manufacturer's instructions and analyzed 48 h later. For Ba/F3 cell infection, retroviral supernatants were obtained as previously described See Cells were treated with lysis buffer . Whole cell lysates (50 µg) or anti-phosphotyrosine immunoprecipitates  (500 µg) were resolved by SDS-PAGE under reducing conditions and blotted with the following antibodies (Ab's): anti-JAK2, anti-p-STAT5 , anti-STAT5, anti- IκBα , anti-Bcl-xL (BD Biosciences), anti-tubulin (Sigma-Aldrich), anti-TBP  and secondary HRP-conjugated anti-mouse/anti-rabbit antibodies . Enriched nuclear and cytoplasmic extracts were obtained using an NE-PER extraction reagents kit  after 10 min of cold hypotonic shock in 10 mM NaCl. Western blots were visualized using SuperSignal West Pico Chemiluminescent Reagent (Thermo Scientific). Densitometry was performed using Image-Gauge v3.46 software .See 6 cells/ml. Staining with annexin V-phycoerythrin (PE) and 7-AAD was performed following the manufacturer's instructions (BD Biosciences). Samples were analyzed through flow cytometry using an EPICS XL cytometer .Apoptosis was measured by the binding of Annexin-V and the incorporation of 7-amino-actinomycin D (7-AAD) after treatment with the inhibitors. Cells were washed twice with cold PBS and then resuspended in 1X Binding Buffer (BD Biosciences) at a concentration of 1×10nu/nu mice  were subcutaneously injected with 107 Ba/F3 cells expressing BCR-JAK2 together with EGFP (right flank) and 107 Ba/F3-mock cells expressing only EGFP (left flank). Mice were examined by palpation for tumor formation for up to 20 days and then sacrificed by cervical dislocation and analyzed in a 2000-MM Image Station  to detect an EGFP signal. Tumors were extracted and disaggregated with 20 µg/ml of collagenase IMDM (Sigma-Aldrich) for 4 h prior to RNA extraction or flow cytometry analysis in an EPICS XL (Beckman Coulter).Ten female BALB/c OlaHsd-Foxn1BCR-ABL using RNA from bone marrow and peripheral blood samples at diagnosis. The result for the M-BCR-ABL breakpoint region was negative (data not shown), but for m-BCR-ABL the reaction revealed a smaller product compared with the positive control  (BCR-JAK2 fusion gene. RT-PCR assays were performed on the same samples (BCR-JAK2 cDNA was amplified using primers flanking the start codon of BCR (BCR-Fw-T) and the stop codon of JAK2 (JAK2-Rv-T). A specific band of 2,260-bp was obtained (BCR followed by the TK domain of JAK2 (JH1) (Standard RT-PCR was performed to detect  control . Sequencr (ABL3) . In line samples  with pri samples . A speciobtained , sequencK2 (JH1) .BCR gene region, 3 signals were detected in 73 of 100 nuclei scored, suggesting that one BCR allele is broken  nuclei scored . The patient acquired CMR five weeks after induction therapy and prior to SCT, and CMR was maintained to the present time (more than six years)  .BCR-JAK2 cDNA (Ba/F3-BCR-JAK2) or the control vector (Ba/F3-mock). As a consequence of BCR-JAK2 expression, IL-3-independent Ba/F3 cells were obtained after 2–4 days of culture, yet no independent cells were obtained with the control vector. Interestingly, Ba/F3-BCR-JAK2 cells showed a cluster-forming growth pattern when compared to Ba/F3-mock cells -dependent Ba/F3 cell line was used as a model ck cells . Prolifeck cells . Ba/F3-BTo test whether Ba/F3 cells stably transduced with pLZR-BJ expressed tyrosine-phosphorylated BCR-JAK2 and activated STAT5, cellular lysates were immunoprecipitated with anti-pTyr Ab and immunoblotted with anti-JAK2 to detect pJAK2. A tyrosine-phosphorylated product of approximately 90 kDa corresponding to the chimeric protein was detected in Ba/F3-BCR-JAK2 cells, whereas no such product was seen in Ba/F3-mock cells . TherefoBcl-xL gene expression was performed on transduced Ba/F3 cells  and suppressor of cytokine signaling-2 (Socs2) In addition to 50 = 6 nM) that induces cell cycle arrest and apoptosis in Ba/F3 cells expressing the JAK2V617F mutation (IC50 = 170 nM) and in JAK2V617F-expressing acute myeloid leukemia cells through the inhibition of pJAK2V617F and pSTAT5 Bcl-xL, Osm and Socs2 transcript detection was performed on Ba/F3-mock, Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 cells treated with TG101209 for 12 h.  cells selected from bone marrow of Balb/c mice was performed and in vitro expansion was carried out  while proliferation of control samples decreased after 30 days in culture. These data suggest that besides the proliferative advantage and ability to transform hematopoietic cell lines  PCR product sequence (281 bp) obtained at diagnosis with the BCR-ABL primers used for p190 detection. Green, BCR-B primer sequence; black, BCR (exon 1); red, JAK2 (exon 19); blue, complementary sequence of ABL3 primer used for the first PCR amplification. The open reading frame is maintained. (b) Detailed sequence alignment analysis of reverse ABL3 primer (blue) showing 14 nucleotides (red) complementary to the flanking region of JAK2 sequence shown in (a).BCR-JAK2 breakpoint region sequence. ((TIF)Click here for additional data file.Figure S2BCR-JAK2 is located in the cytoplasm. Immunofluorescence analysis of HEK293T cells transiently transfected with pLZR carrying BCR-JAK2 (HEK293T-BJ) or control vector (HEK293T-mock) analyzed by confocal microscopy. Transfected cells were detected by EGFP expression. JAK2 expression was shown in red only in the cytoplasm of HEK293T-BJ cells. The merged image showed nuclei stained with DAPI (blue), EGFP (green), and anti-JAK2 Ab (red) on HEK293T-BJ transfected cells. Scale bar = 5 µm.(TIF)Click here for additional data file.Figure S3Bcl-xL expression in the samples from the patient at diagnosis and at complete molecular remission (CMR) for BCR-JAK2. Bars represent relative Bcl-xL levels normalized using H3 and results are given as mean ± SD (n = 3).Quantification of (TIF)Click here for additional data file.Figure S4BCR-JAK2 elicited BcL-xL promoter activation. Luciferase assay of HEK293T cells transiently co-transfected with a plasmid bearing the promoter region of BcL-xL tagged to luciferase (pGL2-pmter Bcl-xL 0.6R) together with plasmids coding for either BCR-JAK2 or a constitutively active STAT5. Luciferase activity induction was calculated as firefly/renilla luciferase activities and normalized using control vector-transfected levels. One representative experiment out of three is shown due to the high variation obtained by transient transfection experiments.(TIF)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


Non-contrast T1-mapping using ShMOLLI can serve as a novel CMR diagnostic criterion in patients presenting with suspected acute myocarditis.The accurate diagnosis of acute myocarditis on cardiovascular magnetic resonance imaging (CMR) often requires multiple modalities, including T2-weighted (T2w), early and late gadolinium imaging. T1-mapping is an emerging technique which is also sensitive to acute changes in free water content. We hypothesized that non-contrast T1-mapping using the novel Shortened Modified Look-Locker Inversion Recovery (ShMOLLI) sequence can serve as a new diagnostic criterion for acute myocarditis.We studied 23 patients with suspected acute myocarditis and 17 healthy controls. All patients presented with chest pain and troponin I > 0.04 ug/L. CMR within 7 days included (1) T2w short-TI inversion recovery (STIR);(2) ShMOLLI T1-mapping; and (3) phase-sensitive late gadolinium enhancement (LGE) . Receiver operator characteristics analysis showed excellent diagnostic performance for both methods: the area-under-the-curve for ShMOLLI T1-mapping=0.96 and STIR=0.93  and the University of Oxford Clarendon Fund Scholarship. Dr. Robin Choudhury is a Wellcome Trust Senior Research Fellow in Clinical Science. Stefan Neubauer and Robin Choudhury acknowledge support from the British Heart Foundation Centre of Research Excellence, Oxford.


SCaMC-1L proteins show higher aminoacid substitution rates than its closest paralog SCaMC-1. In mouse, SCaMC-1Lexpression is restricted to male germ cells and regulated during spermatogenesisbut unexpectedly its localization is not limited to mitochondrial structures.In mature spermatids SCaMC-1L is detected in the mitochondrial sheath butin previous differentiation stages appears associated to cytosolic granuleswhich colocalize with specific markers of the chromatoid body (CB) in post-meioticround spermatids and inter-mitochondrial cement (IMC) in spermatocytes. Theorigin of this atypical distribution was further investigated by transientexpression in cell lines. Similarly to male germ cells, in addition to mitochondrialand cytosolic distribution, a fraction of SCaMC-1L-expressing COS-7 cellsdisplay cytosolic SCaMC-1L-aggregates which exhibit aggresomal-like featuresas the CB. Our results indicate that different regions of SCaMC-1L hinderits import into mitochondria and this apparently favours the formation ofcytosolic aggregates in COS-7 cells. This mechanism could be also operationalin male germ cells and explain the incorporation of SCaMC-1L into germinalgranules.Mitochondrial carriers (MC) form a highly conserved family involved insolute transport across the inner mitochondrial membrane in eukaryotes. Inmammals, ATP-Mg/Pi carriers, SCaMCs, form the most complex subgroup with fourparalogs, SCaMC-1, -2, -3 and -3L, and several splicing variants. Here, wereport the tissue distribution and subcellular localization of a mammalian-specificSCaMC paralog,  The Mitochondrial carrier (MC) family (SLC25) is a highly represented groupof solute transporters in eukaryotic cells. Members of MC family (MCF) performthe transport of metabolites, nucleotides and cofactors across the inner mitochondrialmembrane  and SCaMCs,the ATP-Mg/Pi carriers Mammalian spermatogenesis is an intricate process of cellular differentiationperformed by numerous testis and/or germ cell specific genes. A number ofultrastructural studies have revealed that mitochondria of germinal cellsundergo changes in their appearance, location and number during spermatogenesis, expressedin male germ cells which represents a new link between mitochondria and germinalgranules. SCaMC-1L is detected in the mitochondrial sheath of mature spermatids,but its intracellular location varies in a stage-specific fashion during spermatogenesis,being present in germinal granules, IMC and CB, in spermatocytes and post-meioticspermatids. By ectopic expression in cell lines we have reproduced this complexdistribution and analysed the mechanisms involved in the formation of cytosolicSCaMC-1L aggregates.4930443G12Rik, whoseproduct showed high similarity, 78% and 75% at the amino acidlevel to human and mouse SCaMC-1, respectively ,encoded a predicted protein of 473 amino acids which displayed four calcium-bindingmotifs at identical positions to those of mouse SCaMC-1 and a carboxyl-terminalhalf similar to the MCF  and synonymous (Ks)nucleotide substitution rates by pairwise comparisons between representativemammalian orthologues using the codeml program implemented in the PAML package Ka/Ksratio is commonly used as indicator of selective constraints on the protein-codinggenes with Ka/Ks = 1, <1 and >1indicating neutral evolution, purifying and positive selection, respectively.Among SCaMC-1 orthologs we found a very low pairwise Ka/Ksratio, 0.055±0.012 (average ± SEM), indicative of a strong purifyingselection. As anticipated, the average pairwise Ka/Ksratio found among SCaMC-1L orthologs was significantly largerthan this of SCaMC-1, 0.392±0.085   whereas that synonymous substitutions, Ks,value appeared only slightly lower among SCaMC-1L, 0.427±0.093,than for SCaMC-1 genes, 0.600±0.131), indicating thatSCaMC-1L appears to be subjected to reduced evolutionary pressure to maintainconserved residues compared to SCaMC-1.Searches in annotated databases failed to detect orthologs in non-mammalianvertebrates but detected p<0.001) . This grSCaMC-1Ltranscripts derived exclusively from testis. Subsequently, we confirmed that SCaMC-1Ltranscripts were successfully amplified from testis and, to lesser levels,from brain mRNA, but not from other tissues  where each stage comprises a mix of cells at certain developmentalsteps .During To determine its cellular distribution along spermatogenesis, we performedimmunofluorescence analysis on squash preparations of seminiferous tubulesas described previously In parallel, SCaMC-1L distribution was analyzed by the drying-down technique The presence of MCs in non-mitochondrial membranes, as peroxisomes or plasmamembrane, is not new When a carboxyl FLAG-tagged SCaMC-1L was expressed in COS-7 cells, onlya fraction of the SCaMC-1L-positive cells, about 20%, showed totalco-localization of SCaMC-1L-staining with that of mitochondrial markers asCOX-I  or MitoTTransfections with varying amounts of the SCaMC-1L-expression vector ruledout that the formation of these extra-mitochondrial granules could be an artefactdue to overexpression . The freIn SCaMC-1L-expressing cells containing cytoplasmic SCaMC-1L granules twomajor patterns were clearly distinguished; i) cells showing small, vesicular,filamentous or cup-shaped aggregates scattered throughout the cytoplasm , arrows,Taken together, our results suggest that ectopic SCaMC-1L are not successfullyimported into mitochondria and it is possible that the excess of unfolded(and possibly unprocessed) cytosolic SCaMC-1L may favour the formation ofsmall cytosolic granules that are later assembled in larger perinuclear aggregates.It should be noted that this mechanism could be also operational in male germcells and to contribute to CB formation during spermatogenesis. Similarly,TDRD4/RNF17, a component of mammalian germinal granules, forms large cytosolicgranules when is transfected in cell lines It has been reported that the CB has aggresome-like features Third, SCaMC-1L perinuclear aggregates as aggresomes were highly enrichedin ubiquitinated proteins. (1–160)-SCaMC-1L, (1–160)-SCaMC-1L expressingcells, about 10%, anti-FLAG signals entirely co-localized with mitochondrialmarkers ((1–160)-SCaMC-1L-positive signals showeda partial overlap with mitochondrial SCaMC-1 -SCaMC-1Lstill appeared in extra-mitochondrial aggregates, both disperse or perinuclear(1–184-huSCaMC-1185–476)conferred a predominantly mitochondrial distribution, while the C-terminalhalf of SCaMC-1L caused a frequent localization in non-mitochondrial aggregates  nucleotide substitution rates, about 4.8 fold, in mammalianSCaMC-1L than in their corresponding SCaMC-1 paralogs. This agrees with theaccelerated divergence commonly observed in one of the copies of duplicatedgene pairs The finding of this fifth SCaMC paralog makes the ATP-Mg/Pi carriers themost represented MC subfamily in mammals. Only the ADP/ATP translocases haveundergone a similar expansion SCaMC-1 related genes, we have used proteinand nucleotide sequences of identified SCaMC-1 genes http://blast.ncbi.nlm.nih.gov),Ensembl (www.ensembl.org)and UCSC (http://genome.ucsc.edu).Searches were carried out using default values with the low complexity filteroff. Likewise, BLASTN were performed in genomic TRACE archives in NCBI. Sequencesderived from the most closely related phylogenetic species were used as queryin each search. Protein homology was determined using the BLASTP algorithm.Exon/intron boundaries were confirmed by application of the Splice site predictionby Neural Network program at BDGP .Finally, sequences were aligned by ClustalW followed by manual adjustments.To estimate non-synonymous (Ka) and synonymous (Ks)nucleotide substitution ratio representative mammalian mRNA sequences of SCaMC-1and SCaMC-1L genes were retrieved from the NCBI database, and then convertedto codon alignments by the PAL2NAL server  using corresponding protein sequence alignments. Ka and Ksvalues were calculated with the codeml program implemented in the PAML package To search for Tissue samples from C57BL/6 mice were used for all analysis. All animalwork performed in this study was carried out in accordance with proceduresapproved in the Directive 86/609/EEC of the European Union with approval ofthe Ethics Committee of the Universidad Autónoma de Madrid. Animalswere sacrificed by cervical dislocation, and the testes from adult mouse weredissected and decapsulated in phosphate buffered saline (PBS). After identificationof the waves of the seminiferous epithelium by transillumination, stage-specificshort tubule segments were cut and processed as described For mouse drying-down preparations, stage-specific segments of seminiferoustubules were isolated and transferred in 20 µl of 100 mM sucrose solutionto a small petri dish. Cells were released from tubules by squeezing carefullywith fine forceps, and suspended by gentle pipetting up and down. The cellsuspension was spread on a slide dipped in the fixing solution, , 0.15% Triton X-100, pH 9.2 in PBS), and slideswere dried for 3 h at 37°C in a highly humidified chamber protected fromlight Spermatozoa collected from dissected caput, corpus and cauda epididymiswere washed in PBS, centrifuged at 3000 g and fixed in 4% PFA in PBSfor 30 min at RT. Then cells were collected by centrifugation and dilutedin PBS for slide preparation. Slides were dried at RT and stored at −80°C.4930443G12Rik gene .For normalization, β-actin sequences were amplified in parallel. The primersand conditions used have been previously described The procedures used for total RNA extractions and first strand cDNA synthesishave been described previously 4930443G12Rikgene. We used two sets of PCR primers to generate overlapping fragments: 5′pair ;and the 3′ pair (forward 5′-CAACCTGCTGAGCATCATACCG-3′and reverse 5′-TTCCACTTCTAGATTAATCCAAAAAGCC-3′).PCR fragments were cloned using pSTBlue1-blunt vector (Novagen) and sequenced.Finally, full length coding sequence was assembled and subcloned into theexpression vector pCMV5 digested with EcoRI, to obtain pCMV5-SCaMC-1L. Theplasmid expressing SCaMC-1LFLAG was generated by using PCR to insertDNA encoding the FLAG epitope preceding the termination codon. The SCaMC-1amino-truncated version, pCMV5-NT(1–160)-SCaMC-1L (aminoacids 161–473), was generated by PCR using a forward primer containinga new initiation codon (underlined) and additional nucleotides according torules for consensus for translational initiation, primer ATGGACATTGTTCGTTTCTGG-3′5′-GACC.The SCaMC-1Like full-length cDNA was synthesized from 3 µg of totalRNA of mouse testis with the Cells-to-cDNA™ II Kit (Ambion) using randomprimers to synthesize the first-strand cDNA. PCR reaction was performed usingspecific primers designed according to the predicted (1–184)-SCaMC-1L(185–473)FLAG)and Chimera-2 (1LN(1–184)-huSCaMC-1(185–477)FLAG)were generated exchanging EcoRI fragments between SCaMC-1L*FLAGand pCMV5-SCaMC-1FLAG. Constructs encoding Chimera-3 (1LN(1–184)-huSCaMC-1(185–362)-1L(363–473)FLAG) and Chimera-4 (1LN(1–163)-huSCaMC-1(364–477)FLAG)proteins were obtained from Chimera-1 and SCaMC-1LFLAG, respectively,exchanging an equivalent BglII-EcoRI fragment with SCaMC-1LFLAGto obtain Chimera-3 and with SCaMC-1FLAG for Chimera-4.To obtain chimeric proteins containing regions of human SCaMC-1 and mouseSCaMC-1L, a novel EcoRI site was created into SCaMC-1L cDNA, SCaMC-1L*,at equivalent position to that present in human SCaMC-1 cDNA by PCR, thischange results in a silence mutation. Chimera-1  were used toexpress amino acids 2–162 fused to a 6-histidine tag at the N terminus.Procedures for overexpression and purification of the recombinant proteinwere as described previously 2 atmosphere. For transfections, 25,000cells grown on coverslips were transiently transfected with 0.5 µg ofDNA, except when noted, using the LipofectAMINE reagent as described COS-7 cells obtained from ATCC  were cultured in Dulbecco'smodified Eagle's medium supplemented with 5% inactivated FBS (Invitrogen)at 37°C in a 5% CO2, 1 mM EDTA, 1mM EGTA, 1 mM dithiothreitol pH 7.4, 1 mM iodoacetate and 1 mM phenylmethylsulfonylfluoride). Homogenates were centrifuged at 500 g for 15 min, and the resultingsupernatants were further centrifuged at 9000 g for 15 min to obtain mitochondria-enrichedfractions, which were resuspended in buffer A. Protein determination was performedaccording to the Bradford method. Total protein extraction from cultured cellswas performed as described Testes were collected from mice of different ages and processed in parallel.Samples were homogenized in a dounce homogenizer in 0.8 ml of buffer A  suggesting that SCaMC-1L genes evolved fasterthan SCaMC-1.(TIF)Click here for additional data file.Figure S2Expression of SCaMC-1L throughout the spermatogenic stages.The germ cells present at each stage (I–XII) of the mouse spermatogeniccycle from intermediate spermatogonia (In) to mature sperm are pictured (adaptedfrom (TIF)Click here for additional data file.Figure S3The C-terminal does not affect the intracellular patterns of ScaMC-1L.(A) Representative images of SCaMC-1L-expressing COS-7 cellsshowing the different intracellular patterns observed; cytosolic (a), mitochondrial(b) and extra-mitochondrial aggregates (c). COS-7 cells were transfected witha full-length SCaMC-1L construct unchanged at C-end, co-localization withmitochondrial structures was determined by co-staining with anti-SCaMC-1Land anti-COX-I as mitochondrial control. (B) Representative imagesof COS-7 cells expressing a SCaMC-1L-EGFP fusion protein. SCaMC-1L-EGFP constructwas obtained subcloning the entire mouse SCaMC-1L codingsequence (amino acids 1–473) into pEGFP-N1. SCaMC-1L-EGFP expressingcells were fixed and co-localization with mitochondrial COX-I was determined.Cells showing cytosolic (a), mitochondrial (a) and extra-mitochondrial aggregates(b) are shown. Magnification 63×; scale bar, 10 µm.(TIF)Click here for additional data file.Figure S4Perinuclear honeycomb-like SCaMC-1L aggregates. A representativeimage of SCaMC-1L-transfected COS-7 cells containing SCaMC-1L honeycomb-likeaggregates is shown. SCaMC-1L was detected with specific anti-SCaMC-1L antibodyvisualized with a FITC-conjugated secondary antibody, mitochondrial structuresare stained with anti-COX-I antibody visualized with Cy3-conjugated secondaryantibody; the corresponding merged panel is also shown. Magnification 63×;scale bar, 20 µm. Enlarged image (400×) of the indicated insetis also shown.(TIF)Click here for additional data file.Figure S5Cytosolic SCaMC-1L granules do not co-localize with late endosomal/lysosomalmarkers. COS-7 cells were transiently transfected with FLAG-taggedSCaMC-1L and 24–30 hours later co-localization was analyzed by co-stainingwith specific markers. COS-7 cells were co-stained with anti-SCaMC-1L antibody and specific markers for late endosomes (A) and lysosomes (B). As lateendosomal marker a monoclonal anti-human CD63 antibody  was used at 1∶200. To analyze co-localizationwith lysosomes, cells were stained with 50 mM LysoTracker (Molecular Probes)for 30 min at 37°C prior to fixation. Nuclei were stained with Hoechst.Magnification 63×; scale bar, 20 µm.(TIF)Click here for additional data file.Figure S6Alignment of SCaMC-1 and SCaMC-1L sequences encoded by exons 2 to7 was performed with ClustalW program and coloured using BOXSHADE program.(www.ch.embnet.org).(TIF)Click here for additional data file.Table S1Accession numbers of annotated SCaMC-1 and SCaMC-1L proteins..(DOCX)Click here for additional data file.Table S2SCaMC-1 and SCaMC-1L non-annotated protein sequences used in thisstudy. .(DOCX)Click here for additional data file.MovieS1COS-7 cells were grown on glass plates and transiently transfectedwith SCaMC-1L-EGFP construct. The SCaMC-1L-EGFP expressing vector wasobtained subcloning the full coding sequence (amino acids1–473) of mouseSCaMC-1L-1L into pEGFP-N. Images were taken every 15 min for 7 h using anAxiovert 200 (Zeiss) system at 37 C with a ×40 objective using a CoolsnapFX CCD camera (Roper Scientific). The Metamorph 6.1r6 program was used for image processing.(AVI)Click here for additional data file.


Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.The DAF-16/FoxO transcription factor controls growth, metabolism and aging in  Caenorhabditis elegans) mutation of the insulin/IGF-1 receptor daf-2 dramatically extends lifespan. This is due to the increased activity of DAF-16, a FoxO transcription factor, leading to altered expression of genes, many encoding other regulatory proteins. We have focused on one such protein, AMP-activated protein kinase (AMPK), that is important for regulating cellular homeostasis under conditions of low energy availability (e.g. starvation). We find that DAF-16 binds to the promoter of aakg-4  and increases its expression. Inhibition of aakg-4 leads to down-regulation of multiple DAF-16 target genes and shortens the life of daf-2 mutants. Taken together with a previous report showing that AMPK activates DAF-16, this suggests that AAKG-4 and DAF-16 are involved in a positive feedback loop which accelerates effects of DAF-16 on gene expression, and could contribute to longevity. This study defines a new part of the complex gene regulatory network in which DAF-16 acts to control aging. FoxO-AMPK interactions are present in higher animals, where they could potentially also influence aging.Aging is an important problem for human health and is regulated by complex gene regulatory networks. In a simple nematode worm ( Caenorhabditis elegans, it has long been known that reduction of insulin/IGF-1 signaling (IIS) increases lifespan daf-2 mutants to promote longevity is the FoxO transcription factor DAF-16 In One approach to investigate DAF-16 function is to define the topology of the gene regulatory network within which it acts. To this end we recently used new chromatin profiling data, cross-referenced to mRNA profile data, to identify with high confidence 65 genes subject to direct transcriptional activation by DAF-16 C. elegans there are multiple isoforms of each subunit, aak-1 and aak-2 (α), aakb-1 and aakb-2 (β), and aakg-1-5 (γ) (WormBase version WS238). Mammalian AMPK is activated by binding of AMP or ADP to the γ subunit AMPK acts as a fuel gauge within cells: when the AMP∶ATP ratio rises as energy availability drops, activation of AMPK increases catabolism and reduces biosynthesis C. elegans: The increased lifespan seen in mutants with reduced IIS (e.g. daf-2) or TOR (e.g. rsks-1) pathways, as well as those with mutations affecting mitochondrial function (e.g. isp-1) are aak-2 (i.e. AMPK) dependent Drosophila activation of AMPK by modulating enzymes involved in AMP biosynthesis extends lifespan lkb1 (an upstream activator of AMPK) AMPK inhibits aging in daf-2 longevity requires aak-2, the catalytic subunit of AMPK, may reflect regulation of AMPK by IIS, but the nature of such regulation remains poorly defined aakg-4 and aakg-5, and mRNA profile data suggests that DAF-16 also activates expression of aak-2 and aakb-1aakg-4 subunit is a direct target of DAF-16, leading to increased aakg-4 gene expression. Moreover, reducing aakg-4 expression attenuates daf-2 mutant longevity, implying that this regulation contributes to longevity. Effects of aakg-4 and daf-2 on expression of DAF-16 target genes support the existence of an AMPK-DAF-16 positive feedback loop promoting diapause and longevity. In addition, the daf-16 gene itself contains strong DAF-16 binding sites That aakg-4 and aakg-5, are direct targets of, and transcriptionally activated by DAF-16 C. elegans is unusual in possessing five AMPK γ isoforms, compared to one in DrosophilaC. elegans AMPK γ isoforms but over-expression of a constitutively active form of aakg-2 (R81Q) can modestly increase worm lifespan Our initial mRNA and chromatin profiling analysis suggested that two AMPK γ subunits, aakg-1 is the most similar, followed by aakg-2 and aakg-3  isoform highlights conserved regions corresponding to the mammalian AMPK cystathione-β-synthase (CBS) domains. These domains mediate AMP/ADP and ATP binding to the enzyme allowing AMPK to respond to changes in the energy status of the cell. Interestingly, there is much less CBS domain conservation in aakg-4 and aakg-5 , in which one would expect all AMPK to be AMP/ADP sensitive, with aakg-1/2(RNAi) worms in which one would expect most AMPK to be AMP/ADP insensitive. To test AMPK activity, we measured levels of AMPKα phosphorylation, either in control, nutritionally replete animals, or in ATP-depleted animals (treated with the respiratory inhibitor sodium azide). As predicted, RNAi control animals treated with azide showed increased AMPK activity (aakg-4/5(RNAi) worms but not aakg-1/2(RNAi) worms (aakg-4/5(RNAi) but not aakg-1/2(RNAi)  worms . In nutr/2(RNAi) . These rC. elegans aakg subunits, and the extent of the existence of atypical AAKG isoforms in other species, we identified and analyzed AAKG protein sequences from a range of nematode species. Orthologs of aakg-4 and aakg-5 were found in the nematodes Pristionchus pacificus (order: Rhabditida) and Brugia malayi (order: Spirurida) (Trichuris muris), or among platyhelminth species  , demonst species  and S3. pirurida . The finparalogs , but Spit aakg-3 .C. elegans aakg-4 and -5 also lack key AMP-binding residues   , while takg mRNA . A countant mRNA . Thus, tp<0.001) increases in aakg-4 and aakg-5 mRNA, respectively, in glp-4(bn2); daf-2(m577) relative to daf-16(mgDf50) glp-4; daf-2 young adult hermaphrodites aakg-4 in an additional two profiles aakg-5 in one profile daf-2(e1370) and daf-16; daf-2 young adults. This confirmed that aakg-4 mRNA levels are elevated in daf-2 relative to daf-16; daf-2, but for aakg-5 this could not be confirmed  and daf-16; daf-2 adults using chromatin immunoprecipitation (ChIP) with a DAF-16-specific antibody (Santa Cruz)  (aakg-4 peak or that of aakg-5 (aakg-4 is a bona fide DAF-16 target gene and DAF-16 binding to the aakg-5 promoter is either non-existent or condition dependent.Next we verified binding of DAF-16 to the promoters of ta Cruz)  and PCR.h aakg-5  and we e(peak 2) . Howeverf aakg-5 . Thus, iaakg-4 and aakg-5, and their regulation by IIS and DAF-16, we created transgenic lines containing Paakg-4::gfp and Paakg-5::gfp transcriptional reporters. Paakg-4::gfp expression was observed in many tissues, particularly the intestine, body wall muscle, vulval muscles and several neurons in the head and tail including amphids and phasmids, respectively  and daf-16; daf-2 backgrounds and GFP fluorescence measured. Consistent with aakg-4 mRNA data, Paakg-4::gfp expression was increased 1.6 fold by daf-2, and this increase was daf-16 dependent  C. briggsae aakg-4 promoter, suggesting conservation of regulation by IIS/FoxO, but not in C. remanei. DBEs were not detected in the aakg-5 promoter of any of these species. These findings strongly imply that DAF-16 binds to the promoter of aakg-4 and activates its expression.We also examined the glp-4; daf-2(m577) relative to daf-16; glp-4; daf-2 young adult hermaphrodites aak-1 (α1 subunit) and aakb-2 (β2) mRNA levels were not different between the two strains, but aak-2 (α2) and aakb-1 (β1) were both elevated in daf-2 vs daf-16; daf-2, showing 2.28-fold and 7.51-fold (both p<0.001) higher levels, respectively, in daf-2. aakb-1 activation by DAF-16 was also implied by two other mRNA profile studies aakb-1 mRNA levels were also higher in daf-2(e1370) relative to N2 in individually profiled 19 day old worms aak-2 mRNA levels were not seen in any of these other studies. For verification, we then used qRT-PCR to compare aak-2 and aakb-1 mRNA levels in 1 day old daf-2(e1370) and daf-16; daf-2 adults. This confirmed a statistically significant increase for aakb-1, but also suggested a possible increase in aak-2 mRNA levels . To test this the deletion mutation aakb-1(tm2658) was combined with daf-2(m577), and lifespan measured at 25°C. aakb-1 proved to shorten lifespan in daf-2(m577) and wild type worms to a similar degree   on combined data for all trials, but no difference was detected (p = 0.25). This suggests that activation of aakb-1 expression does not contribute to daf-2 Age.The evidence of up-regulation of  trials) . To estiaakg-4 contributes to daf-2 Age, we obtained the mutation aakg-4(tm5539). This deletes 405 bp from the gene, in a region encoding portions of both CBS3 and CBS4, and is therefore predicted to be a null. aakg-4 mutants appeared healthy and showed normal fertility (aakg-4 reduced lifespan in a daf-2(m577) background , but not in a daf-2(+) background (aakg-4 effects on lifespan were greater in a daf-2 background (CPHA p<0.0001), implying that aakg-4 contributes to daf-2 Age. To confirm this, we abrogated aakg-4 expression using RNAi, and again observed a greater reduction in lifespan in daf-2 than in wild-type worms  (To assess whether ertility . aakg-4 ckground . Statist 0.0024) .aakg-5 also contributed to daf-2 Age we used aakg-5(RNAi). Like aakg-4(RNAi), this too markedly reduced lifespan in daf-2 but not N2   . We alsoaf-2 Age .aakg-4 and aakg-5 form a distinct clade within the C. elegans AMPK γ subunits  could further reduce daf-2 Age in daf-2;aakg-4 worms, but it could not .Taken together, these findings imply that in  AMP/ADP  suggestsdaf-16-dependent up-regulation of expression of aakg-4, and possibly also aak-2 and aakb-1, in daf-2 mutants. It was shown previously that AMPK directly phosphorylates and activates DAF-16 aakg-4 should promote DAF-16 activity. To test this we used expression of DAF-16 target genes as a readout of DAF-16 activity.We have identified sod-3 is induced by reduced IIS and has also been shown to be regulated by aak-2sod-3 expression can be induced by shifting rrf-3; daf-2(m577) worms from 15°C to 25°C   was initiated at the same time as the temperature shift there was a significant reduction in sod-3 expression after 24 hr in animals treated with aakg-4(RNAi)  . The rrf animals . Notably-4(RNAi) . Moreoveaakg-4(RNAi) in rrf-3; daf-2(m577) worms on expression of seven other known DAF-16 target genes aakg-1, aakg-2 and aakg-5 RNAi on sod-3 expression but could detect no significant effect  in daf-2 mutant animals reduced nuclear localization of DAF-16::GFP, but only marginal effects were detected  mutants in the presence of the daf-16(m26) point mutation. No difference was detected, implying that daf-16 does not activate its own expression  worms, predicted to contain only AMP-dependent AMPK, but not in aakg-1/2(RNAi) worms, predicted to contain only Chromadorean AMPK  but not aakg-2/-3(RNAi) did show slightly reduced pAMPKα levels in unstressed, fully fed worms  is particularly important in cardiac function The presence of two α, two β and five γ AMPK subunits in of aak-2 . Both dantestine . If the aak-2 can act with either aakb-1 or aakb-2 and both β subunits need to be removed to fully abrogate the long lifespan of dauer larvae aakb-1 mutation alone does not affect daf-2 adult lifespan  delays induction of the DAF-16 target gene sod-3 (aakg-4(RNAi) was transient i.e. detected after 24 hr but not 48 hr , GA303 rrf-3(pk1426); daf-2(m577), GA1073 rrf-3(pk1426); daf-16(mgDf50); daf-2(m577), GA1061 aakb-1(tm2658) , GA1069 daf-2(m577); aakb-1(tm2658), GA1071 aakg-4(tm5539) , GA1072 daf-2(m577); aakg-4(tm5539), GA1052 sEx10615 [Paak-2::gfp], GA1048 daf-2(e1370) sEx10615 [Paak-2::gfp], GA1050 daf-16(mgDf50); daf-2(e1370) sEx10615 [Paak-2::gfp], GA1051 sEx11830 [Paakb-1::gfp], GA1047 daf-2(e1370) sEx11830 [Paakb-1::gfp], GA1049 daf-16(mgDf50); daf-2(e1370) sEx11830 [Paakb-1::gfp], GA1403 wuEx256 [Paakg-4::gfp], GA1404 daf-2(e1370) wuEx256 [Paakg-4::gfp], GA1405 daf-16(mgDf50); daf-2(e1370) wuEx256 [Paakg-4::gfp], GA1410 wuEx257 [Paakg-4::gfp], GA1411 daf-2(e1370) wuEx257 [Paakg-4::gfp], GA1412 daf-16(mgDf50); daf-2(e1370) wuEx257 [Paakg-4::GFP], GA1400 wuEx251 [Paakg-5::gfp], GA1401 daf-2(e1370) wuEx251 [Paakg-5::gfp], GA1402 daf-16(mgDf50); daf-2(e1370) wuEx251 [Paakg-5::gfp], GA1413 wuEx258 [Paakg-5::gfp], GA1414 daf-2(e1370) wuEx258 [Paakg-5::gfp], GA1415 wuEx258 daf-16(mgDf50); daf-2(e1370) [Paakg-5::gfp], GA1431 wuEx261 [Pdaf-16a(i)::gfp], GA1433 daf-2(e1370) wuEx261 [Pdaf-16a(i)::gfp], GA1435 daf-16(mgDf50); daf-2(e1370) wuEx261 [Pdaf-16a(i)::gfp], GA1419 wuEx260 [Pdaf-16a(i)::gfp], GA1420 daf-2(e1370) wuEx260 [Pdaf-16a(i)::gfp], GA1421 daf-16(mgDf50); daf-2(e1370) wuEx260 [Pdaf-16a(i)::gfp], GA1437 wuEx263 [Pdaf-16a(ii)::gfp], GA1440 wuEx263 [Pdaf-16a(ii)::gfp], GA1442 daf-2(e1370) wuEx263 [Pdaf-16a(ii)::gfp], GA1438 daf-16(mgDf50); daf-2(e1370) wuEx264 [Pdaf-16a(ii)::gfp], GA1441 daf-2(e1370) wuEx264 [Pdaf-16a(ii)::gfp], GA1443 daf-16(mgDf50); daf-2(e1370) wuEx264 [Pdaf-16a(ii)::gfp].Worms were maintained at 20°C unless otherwise indicated. The following strains were used: N2, NL2099 aakb-1(tm2658), was carried out by lysis of parent animals using proteinase K (Sigma) and subsequent PCR using the following primers. F:gcaatgtgattaaaagttatggg, In:cgatgataatcagcaaaagacg, R:cagggtatactacacatgtacc. Primers for aakg-4(tm5539) were F: agtctctgacacgccgagtt, In: gcacaggcttttagacttcg, R: cgaagtctaaaagcctgtgc.Multiple mutants were created using standard methodologies and the presence of genomic deletions was tested either by screening for characteristic phenotypes or via PCR. In the case of PCR, genotyping, of promoter::gfp transgenes of daf-16a were created using Gateway cloning (Invitrogen). The primer sequences that define the region used for Pdaf-16a(i) were: F:cctccatcaacaagagcgttc and R:gcaaatgcaacacggagaaaacg; and for the Pdaf-16a(ii): F:catgtctcgtgtgcctctcctttcca and R:taacgtcttcgggaatttcagccaaag. The sequences used to define the daf-16 3′UTR were F:attctcttcattttgtttcccctggtgttgttcg and R:catcatcatacagtcgcaaatatatttggggg. The promoter::gfp fusions for aakg-4 and aakg-5 were made as previously described unc-54 3′UTR. The sequences that define the promoter used for Paakg-4 were F:aaaagacacactcaatttccataaatatat and R:tgataaatgatgatattttgaggttgtgaaa and for Paakg-5 F:agatcaaaggcttattgtgcatttctattt and R:tctggaaaataaaagcattaaagtgaaaaat. In each case the transgenic strains were created by microinjection of the plasmid (Pdaf-16) or PCR product (Paakg-4 and 5).The aakg-1, aakg-2 and aakg-4 RNAi clones were obtained from the Ahringer library and those for aakg-3 and aakg-5 were constructed de novo. The primer sequences defining the regions used for aakg-3 were: F:attaaatgcggccgctaaggataggagttcgtaagtatcaattag and R:attaaagacgtcgtaatatcctacgatgtgtcaatatgtacg; and for aakg-5 F:attaaagtcgacacacaccagcatccgaacaacgtcgtaact and R:attaaagagctcctaaagattcttcagcttgctgcagacatt. The primers contain NotI, PstI, SalI and SacI restriction sites, respectively, allowing the resulting PCR fragments to be cloned into pL4440.E. coli HT115 transformed with RNAi plasmids. The resulting progeny were themselves allowed to develop to young adults at 20°C before harvesting in M9 buffer. Worms were then placed either on control plates or plates containing 1 mM sodium azide for 2 hr at 20°C E. coli OP50 72 hr before use.Adult worms were allowed to lay eggs for 12 hr on plates seeded with Following treatment worms were washed off plates using M9, collected by settling and resuspended in 100 µl Phosphosafe extraction reagent (Millipore) supplemented with a protease inhibitor cocktail (Roche). Protein concentration was determined using BCA (Pierce) and 40 µg protein was separated by SDS-gel electrophoresis. Proteins were subsequently transferred to nitrocellulose membrane and probed using a 1∶1000 dilution of phospho-AMPKα (Thr172) antibody . β-actin antibody (Santa Cruz) was used as a loading control. Imaging and quantification of bands was carried out using the ImageQuant LAS4000 imaging system and software .daf-2 phenotype in the daf-2(m577) 1 day old adults. For each slide, 30–40 worms were mounted in M9+0.2% levamisole on a 2% agarose pad and imaged within the following 30 min. Quantification of GFP expression from transgenic strains was carried out using a Leica DMRXA2 microscope using a GFP filter cube , an Orca C10600 digital camera (Hamamatsu) and Volocity image analysis software (Improvision). Low level intestinal autofluorescence was detected in the GFP range in all our strains. For the highly expressed Paakg-4::gfp and Paakg-5::gfp reporters this did not interfere with our analysis but for Paak-2::gfp and Paakb-1::gfp which required longer exposure times we corrected for autofluorescence using GFP measurements from non-transgenic animals or by measuring GFP levels only in the head where autofluorescence is minimal.Worms were raised at 15°C, picked at L4 stage, and shifted to 25°C for 24 hr to increase transgenic extrachromosomal array expression and to induce the Confocal images were acquired using the Zen2009 software driving a LSM-710 confocal station with an inverted Axio Observer microscope . Whole worm images were generated from the Z-projection of 12–18 XY-planes acquired every 3 µm through a 10× Plan-Apochromat 0.45 NA dry objective . High-resolution images were generated from the Z-projection of 45–60 XY-planes acquired every 0.75 µm through a 40× Plan-Apochromat 1.3 NA oil objective . Z-projections were created using the freeware LSM5 image browser . Tiff images were then exported to Photoshop CS4 (Adobe), background was subtracted and contrast adjusted for optimal display.E. coli. 50–100 worms were used for each assay. RNA was extracted using Trizol (Sigma) and cDNA synthesized using SuperScript II reverse transcriptase with oligo dT (Invitrogen). qRT-PCR was carried out using Fast SYBR Green Master Mix (Applied Biosystems) and the 7900 HT Fast PCR system (Applied Biosystems). Normalization of transcript quantity was carried out using the geometric mean of three stably expressed reference genes Y45F10D.4, pmp-3, and cdc-42 in order to control for cDNA input, as previously described aakg-4 (peak 1) F:tctcacactcccttcccact and R:gccgtcgtcacaaatactga; aakg-4 (peak 2) F: aaaaagcgagcaaagcaaaa R: ttccacatttgtcgcacttc; and aakg-5 (peak 1 primers 1) F:aggacggactgttttgttgc and R:gctcctcgttttcaatgctt. The relative levels of aakg isoform mRNA were determined using custom made primers designed and synthesized by Primerdesign. These were optimized to amplify each of the 5 subunits at the same rate, thus allowing relative quantification without the need for an internal standard.RNA was isolated from adult worms after transfer of the worms to an unseeded NGM plate to remove C. elegans cultures were grown on plates on OP50 at 20°C. The worms were collected, washed 4× in PBS buffer, frozen in liquid nitrogen and pulverized with a mortar and pestle. The resulting powder was then re-suspended in 8 ml PBS containing 1% formaldehyde and incubated for 20 min with gentle mixing at room temperature. Crosslinking was stopped by addition of 400 µl 2.5 M glycine solution and 20 min further incubation at room temperature. After 4 washes in PBS containing EDTA-free protease inhibitor tablets , samples were flash frozen and stored at −80°C. After thawing, 2 ml of HLB buffer  was added and sonication was carried out at 70% intensity for 7 bursts of 30 s in a Vibracell sonicator (Sonics). Protein quantity was estimated by BCA (Pierce) and 2 mg were diluted to 500 µl in HLB buffer.The protocol for chromatin immunoprecipitation was adapted from a previous report 3×50 µl aliquots were removed at this point. DNA isolated from these samples was subsequently used for input controls. Samples were pre-cleared for 1 hr in prewashed salmon sperm DNA/protein-A agarose beads (Millipore) and then incubated overnight with 10 µl of anti-DAF-16 Ab (Santa Cruz). Samples were then incubated with prewashed salmon sperm DNA/protein-A agarose beads for 2 hr. The beads were then washed twice in WB1 , twice in WB2  and once in WB3 . Crosslinking was reversed by addition of proteinase K solution  and incubation for 2 hr at 45°C and overnight at 65°C. DNA was isolated by applying solution to Qiagen PCR purification columns.p values (log rank test) were determined using JMP software, version 7. Cox proportional hazard p values were determined in R from combined experiments using mixed effects models . For lifespans using RNAi worms were grown on bacteria expressing aakg RNAi clones from the L4 stage. Empty pL4440 vector was used as a control.Prior to experiments animals were maintained at the permissive temperature and grown for at least one generation in the presence of food to assure full viability. Lifespan assays were performed essentially as described C. elegans genes aakg-1-5 were identified by local alignment searches of the five protein sequences to the gene models in WormBase. Since not all genomes were available in WormBase orthologs for the parasitic helminths  were derived from GeneDB. Orthologs for G. pallida, H. contortus, O. volvulus, S. ratti and T. muris were derived using data available on the Sanger Institute Scientific Resources website. Since there are no gene model predictions available for O. volvulus and T. muris, we identified contigs that aligned closely to the C. elegans gene sequences of interest using a “protein versus translated DNA” Blast search. We then predicted genes within these contigs using the Eukaryotic GeneMark.hmm C. elegans aakg genes. Finally, orthologs of P. pacificus were derived from the gene predictions available from www.pristionchus.org.For the majority of species orthologs of the Multiple sequence alignments of the protein sequences were done using MUSCLE Figure S1C. elegans and B. malayi. It is possible that the presence of atypical AAKG isoforms in the Enoplid nematodes T. muris and T. spiralis reflects the existence of a primordial atypical isoform in the common ancestor of the Chromadorea and Enoplea. The earlier existence of this atypical isoform is unclear: Parasitic platyhelminthes contain a divergent AAKG isoform in which the AMP-binding residues are largely conserved  that clusters with the AAKG-4/5 group, but this is exceptional Click here for additional data file.Figure S2C. japonica, which lacks an aakg-5 ortholog.Relationship between γ isoforms in the Rhabditida. Representatives of the 5 AAKG isoforms are identifiable in most Rhabditid species. An exception is (PDF)Click here for additional data file.Figure S3Evolutionary relationship between nematode and platyhelminth lineages. Species shown are represented in (PDF)Click here for additional data file.Figure S4Immuno-precipitation and Western blot with anti-DAF-16 antibody. Immuno-precipitation was carried out as for ChIP using the anti-DAF-16 ce-300 antibody . The resulting lysates were separated on an SDS gel and transferred to nitrocellulose membrane before probing with a second DAF-16 antibody raised in a different species to avoid cross-reactivity .(PDF)Click here for additional data file.Figure S5aak-2 or aakb-1. In that study to avoid false positives we applied strict criteria to define DAF-16 binding sites, including location no more than 1 Kb from the predicted translational start site. However, a potential DAF-16 binding site is present in the aakb-1 promoter 1.5 Kb from the start site, in the vicinity of a weak DAF-16 binding element (DBE) aak-2 promoter.DAF-16 binding profiles in four AMPK genes. Peaks represent sites of increased DNA methylation caused by binding of DAF-16::Dam methylase fusion protein (DamID). Plots generated using data from a previous study (PDF)Click here for additional data file.Figure S6aakg-5 is broadly expressed but its transcription is not regulated by IIS. A) Confocal images showing the Paakg-5::GFP expression pattern in 1-day old hermaphrodites. The Paakg-5::GFP transgene was created using the fusion method of PCR to ‘stitch’ together 1.85 Kb of promoter taken directly upstream of the transcriptional start site, GFP and the unc-54 3′UTR Paakg-5::GFP is expressed in the female gonad sheath cells (GonSh), vulva epithelium (VlvE) and neurons (VlvN), ventral cord neurons (VC) and excretory cell (Exc). (iii) It is also seen in the spermatheca (Sperm) and epithelial seam cells (Seam). (iv) In addition to the excretory cell (Exc), Paakg-5::GFP displays strong expression levels in the pharyngeal epithelia (PhxE), neurons (PhxN), some ring neurons (RingN) and sensory neuron (SensN) termini. (v) In the tail, Paakg-5::GFP signal mostly localizes to the pre-anal ganglion (RectN), rectum epithelium (RectE), intestinal-rectal valve (RectV) and phasmid support cells . As seen along the whole worm, it is also clearly expressed in the seam cells (Seam), intestine (Int) and excretory cell arms (Exc). Paakg-5::gfp with other AMPK subunits. B) Quantification of GFP fluorescence in worms expressing a Paakg-5::GFP reporter. We did not observe any differences in GFP fluorescence levels when our transgene was crossed into daf-2 or daf-16; daf-2 backgrounds. The expression pattern was also unchanged (data not shown). The same was also true for a second set of strains generated from a different extrachromosomal array. Error bars, standard deviation. C) qRT-PCR of gfp mRNA in worms expressing a Paakg-5::GFP reporter did not reveal any change in expression between daf-2 and daf-2; daf-16. Error bars, standard deviation.(PDF)Click here for additional data file.Figure S7aakb-1 by insulin/IGF-1 signaling. qRT-PCR data. A, B) aakb-1 but not aak-2 mRNA levels are increased in daf-2 animals in a daf-16-dependent manner. ♦p<0.08 compared to N2, *p<0.08 compared to daf-2. Error bars, standard deviation.Evidence of regulation of (PDF)Click here for additional data file.Figure S8aak-2 by insulin/IGF-1 signaling. A) Confocal images showing Paak-2::GFP expression pattern in 1-day old wild type hermaphrodites. The Paak-2::gfp sequence in sEx10615 contains 2.821 Kb of DNA upstream of the transcriptional start site. (i) Whole worm expression pattern. (ii) Head Paak-2::GFP expression is mostly seen in the excretory cell (Exc) but is also observed in pharyngeal neurons (PhxN), epithelial cells (PhxE), a subset of ring neurons (RingN), amphid socket cells (AmphSc) and head body wall muscles (HM). (iii) Paak-2::GFP is also expressed in the posterior intestine (Int), rectal gland (RectG) and epithelial cells (RectE), and in phasmids (Pha). (iv) Paak-2::GFP is detected in vulval muscles (VlvM). This expression pattern builds on but is consistent with previous observations of this strain Paak-2::aak-2::gfp translational reporter. Paak-2::gfp with other AMPK subunits. B) Quantification of GFP fluorescence in worms expressing a Paak-2::GFP reporter. We did not observe any differences in GFP fluorescence levels when our transgene was crossed into daf-2 or daf-16; daf-2 backgrounds. The expression pattern was also unchanged (data not shown). GFP fluorescence values are corrected for background intestinal autofluorescence using values obtained from non-transgenic animals (see methods). Error bars, standard deviation. C) qRT-PCR of gfp mRNA in worms expressing a Paak-2::GFP reporter revealed a small, significant change in expression between daf-2 and daf-2; daf-16. ♦p<0.05 compared to N2, *p<0.05 compared to daf-2. Error bars, standard deviation.Evidence of regulation of (PDF)Click here for additional data file.Figure S9aakb-1. A) Confocal images showing Paakb-1::GFP in 1-day old wild type hermaphrodites. The Paakb-1::gfp reporter contains 2.845 Kb of promoter directly upstream of the transcriptional start site. (i) Whole worm expression pattern. (ii) Paakb-1::GFP is strongly expressed in pharyngeal muscles (PhxM), the pharyngeal intestinal valve cells (VPI) as previously noted Paakb-1::GFP includes the tail minor epithelium (TE), the rectal-intestinal valve (RectV), posterior intestine and body wall muscles (BWM). (iv) Mid-body expression is mostly restricted to muscles of the vulva (VlvM), uterus (UtM) and body wall (BMW), but is also detected in the intestine (Int). However we noted that the levels of GFP expression in all genetic backgrounds was highly variable between animals. Paakb-1::gfp with other AMPK subunits. B) Quantification of GFP fluorescence in the heads of worms expressing a Paakb-1::GFP reporter. The daf-16; daf-2 background carrying this transgene exhibited unexplained and higher than normal intestinal autofluorescence making fluorescence quantification of whole worms difficult. To circumvent this we measured GFP fluorescence levels in the heads of worms, where Paakb-1::GFP is highly expressed. Mean of four trials shown. Error bars, standard deviation. C) qRT-PCR of gfp mRNA in worms expressing a Paakb-1::GFP reporter revealed an increase in expression between daf-2 and daf-2; daf-16, however this was not significant, probably due to transgene expression varying between individuals. Error bars, standard deviation.Expression pattern of (PDF)Click here for additional data file.Figure S10aakb-1 does not contribute to daf-2 longevity. The proportional reduction in lifespan caused by aakb-1(tm2658) does not significantly differ between daf-2(+) and daf-2(m577) backgrounds. The combined data from 4 trials is shown (C in own C in .(PDF)Click here for additional data file.Figure S11aakg-4(tm5539) does not alter progeny production. The total progeny were counted from individual adult worms. This graph represents data from two independent trials. n = 20 broods for each genotype, * p<0.05 compared to N2.(PDF)Click here for additional data file.Figure S12aakg-5 RNAi shortens daf-2 lifespan. One representative experiment is shown corresponding to trial 9 in aakg-5 mRNA was confirmed by qRT-PCR (data not shown). However, aakg-5 RNAi did not further reduce the lifespan of daf-2(m577);aakg-4(tm5539) double mutants.(PDF)Click here for additional data file.Figure S13sod-3 in daf-2 mutants. A) L4 daf-2(m577) animals were shifted to 25°C prior to RNA extraction. B) sod-3 mRNA levels are induced in response to temperature specifically in daf-2 mutants.Induction of the DAF-16 target gene (PDF)Click here for additional data file.Figure S14aakg RNAi on induction of sod-3 expression by reduced IIS. A) Experimental protocol. L4 daf-2(m577) animals were treated with aakg RNAi and shifted to 25°C prior to RNA extraction  sod-g-4 RNAi . C) Stat(PDF)Click here for additional data file.Figure S15aakg RNAi on DAF-16::GFP nuclear localization. We created a strain that expressed the DAF-16a::GFP transgene daf-2(e1370) background. daf-2(e1370) is a particularly strong class 2 daf-2 allele, and DAF-16::GFP shows exclusively nuclear localization at 25°C. However, at A) 15°C or B) 20°C , weaker DAF-16::GFP nuclear localization is seen p, Chi Squared test vs control.Effects of (PDF)Click here for additional data file.Figure S16daf-16 mRNA levels are affected in a daf-2 background by daf-16(mgDf50)  but not by daf-16(m26) . **p<0.002, *p = 0.03. B) It remains possible that daf-16 activates its own expression in some worm tissues or cell types, but that this is not detectable in whole worm mRNA extracts. To test this we created and examined Pdaf-16a::gfp transcriptional reporters. Given the large size of the predicted DAF-16-binding region, 2 types of Pdaf-16a::gfp reporters, i and ii, were created containing overlapping 5′ and 3′ regions of the promoter daf-16a::GFP transgenes were designed based on DAF-16 binding sites predicted by DamID and modENCODE. Transgenes were introduced as extrachromosomal arrays into wild-type worms and two independent transgenic lines for each construct were isolated. In a wild-type background, both constructs led to near ubiquitous GFP expression patterns similar to those previously reported for DAF-16a::GFP translational reporters Pdaf-16a(i)::GFP or Pdaf-16a(ii)::GFP reporters. We observed a decrease in GFP fluorescence levels when our transgene was crossed into daf-2 which was reversed by daf-16. The same was also true for a second set of strains generated from different extrachromosomal arrays. The expression pattern was unchanged (data not shown). ♦p<0.01 compared to N2 *p<0.01 compared to daf-2. Error bars, standard deviation. E, F) Quantification of gfp mRNA in worms expressing Pdaf-16a(i)::GFP or Pdaf-16a(ii)::GFP reporters. ♦p<0.01 compared to N2, *p<0.01 compared to daf-2. Error bars, standard deviation. In worms with Pdaf-16a(i)::gfp reporters, GFP fluorescence levels were slightly decreased in daf-2(e1370) compared to daf-16(mgDf50); daf-2 backgrounds, but gfp mRNA levels were not (Pdaf-16a(ii)::gfp reporters, GFP fluorescence and gfp mRNA were both reduced in daf-2 animals compared to daf-16; daf-2  qRT-PCR shows that were not . However6; daf-2 . These r(PDF)Click here for additional data file.Table S1C. elegans AMPK γ subunit CBS domains to human PRKAG1.Percentage amino acid sequence similarity of (PDF)Click here for additional data file.Table S2C. elegans atypical isoforms aakg-4 and aakg-5 Click here for additional data file.Table S3C. elegans atypical aakg-4 and aakg-5 which all contain atypical residues. A number of the γ subunit isoforms from various more distant species contain missing or truncated cystathione-β-synthase (CBS) domains, it is possible that these may be pseudogenes. Alternatively, in AMPK complexes containing these isoforms, enzyme function may be compromised by lacking these CBS domains. However, it is possible that only two CBS domains can come together to form the nucleotide-binding region, as seen in other CBS-domain containing proteins AMP/ATP binding residues in Nematoda and Platyhelminthes. All sequences obtained were aligned simultaneously using ClustalW with an identity matrix. Residues known to be involved in nucleotide binding were identified using the human PRKAG1 gene. Atypical residues are in red. Grey blocks show isoforms that cluster with (PDF)Click here for additional data file.Table S4p values are adjusted and some are not, those in bold are considered significant by the authors. For studies comparing daf-2 with daf-16(mgDf50);daf-2 values are not noted for daf-16 as probe sets fall within the boundaries of the mgDf50 deletion. McElwee et al., 2003: the numbers are an average of four replicates and SD = standard deviation. Budovskaya et al., 2008: negative numbers indicate that the gene is positively regulated by DAF-16. Values represent a total difference across Stanford and Caltech arrays. No differences in aak-1, aakb-1 or aakg-1-3 mRNA levels were detected by any of these studies.Effects of insulin/IGF-1 signaling on AMPK gene expression. Fold changes in mRNA levels are shown. Some (PDF)Click here for additional data file.Table S5aakg-4 peaks and one aakg-5 peak . The values obtained for a positive control DAF-16 target gene M01H9.3Statistical analysis of ChIP data. The % input for three individual trials was compared for the two g-5 peak . aakg-4 (PDF)Click here for additional data file.Table S6aakb-1(tm2658) on lifespan. aakb-1(tm2658) shortens lifespan modestly and to a similar degree in daf-2(+) and daf-2(−) backgrounds. This argues against a role for increased aakb-1 expression as a cause of daf-2 Age. C, data from all four trials combined. N, number of worms . Lifespan assays all carried out at 25°C. p, Probability (determined by log rank test) of being the same as a N2 or bdaf-2(m577). % difference in mean lifespan compared to a N2 or bdaf-2(m577). SEM, standard error of the mean.Effects of (PDF)Click here for additional data file.Table S7aakg-4(tm5539) shortens daf-2 lifespan. N, number of worms . p, Probability (determined by log rank test) of being the same as adaf-2(m577), b N2 or caakg-4(tm5539). % difference in mean lifespan compared to daf-2(m577). SEM, standard error of the mean.(PDF)Click here for additional data file.Table S8aakg-4 and aakg-5 RNAi shorten daf-2 lifespan. N, number of worms . The rrf-3 mutation sensitizes the animals to the effects of RNAi aakg mRNA levels were reduced by aakg-4 or aakg-5(RNAi) (data not shown). In the case of aakg-5 RNAi we sometimes observed an additional decrease in aakg-4 mRNA levels in worms that had been fed RNAi for longer times suggesting that there may be some cross RNAi that could affect lifespan. p, probability (determined by log rank test) of being the same as: arrf-3(pk1426); daf-2(m577) or daf-2(m577) control RNAi. brrf-3(pk1426) or N2 control RNAi. caakg-4(tm5539). ddaf-2; aakg-4(tm5539). % difference in mean lifespan compared to arrf-3(pk1426) or N2. brrf-3(pk1426); daf-2(m577) or daf-2(m577). caakg-4(tm5539). ddaf-2; aakg-4(tm5539). SEM = standard error of the mean. NS, not significant (p>0.05).(PDF)Click here for additional data file.Table S9aakg-1, aakg-2 or aakg-3 does not consistently suppress daf-2 mutant longevity. N, number of worms . Lifespan assays all carried out at 25°C. The rrf-3 mutation sensitizes the animals to the effects of RNAi p, probability (determined by log rank test) of being the same as arrf-3; daf-2 control RNAi or brrf-3 Control RNAi. % differences in mean lifespan are compared to daf-2(m577). SEM: standard error of the mean.RNAi targeted to (PDF)Click here for additional data file.Table S10Paak-2::GFP, Paakb-1::GFP and Paakg-4::GFP expression was observed.Expression pattern of AMPK subunits. Y = expression observed in this tissue. Dark grey boxes highlight tissues where (PDF)Click here for additional data file.