Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS–associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16–dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to  Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode  Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode  Caenorhabditis elegans orthologue of the multifunctional DNA/RNA binding protein TDP-43 (TAR DNA Binding Protein 43). Mutations and accumulations of TDP-43 have been found in patients with Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia, and in a growing number of neurodegenerative disorders TDP-1 is the C. elegans, IIS initiates a phosphorylation cascade through the DAF-2/Insulin-IGF receptor that phosphorylates the forkhead transcription factor DAF-16 and retains it in the cytoplasm daf-2 mutants relieve DAF-16 phosphorylation causing DAF-16 to translocate to the nucleus where it activates a large number of genes resulting in increased lifespan and augmented stress resistance daf-2 mutants are also resistant to numerous stresses including oxidative, osmotic, thermal and proteotoxicity In worms a major axis of stress-response signaling and longevity is the Insulin/IGF-signaling (IIS) pathway. IIS follows an evolutionarily conserved and genetically regulated pathway that regulates numerous processes including development, metabolism, fecundity, cellular stress resistance and aging C. elegans. We asked if TDP-1 participated in the IIS pathway to specify developmental, stress response, and longevity outcomes. Given the importance of human TDP-43 to neurodegeneration we also asked if tdp-1 regulated age-dependent proteotoxicity. Our work points to TDP-1 as a key stress response protein at the crossroads of IIS, proteotoxicity and endoplasmic reticulum (ER) stress. Moreover, persistent induction of TDP-1 may actively promote neurodegeneration.We attempted to address this issue by asking whether TDP-1 participated in the cellular stress response and longevity pathways in tdp-1 participated in the IIS pathway we used a deletion mutant tdp-1(ok803) that removes the two RNA Recognition Motifs of TDP-1 (daf-2(e1370) animals were long-lived but this phenotype was significantly reduced in a daf-2(e1370);tdp-1(ok803) double mutant strain (daf-16(mu86) mutants were short-lived compared to wild type worms and the inclusion of tdp-1(ok803) did not further reduce daf-16 mutants' lifespan (tdp-1 regulated daf-2 mediated longevity with RNA interference and observed that tdp-1(RNAi) reduced the extended lifespan of daf-2 mutants  limiting the extreme lifespan of daf-2 mutants we observed that at 20°C tdp-1(ok803) mutants had a small but significant increase in lifespan versus N2 controls  greatly reduced lifespan compared to wild type N2 worms and tdp-1 deletion mutants at 20°C (tdp-1(ok803);TDP-1::GFP strain was greater than TDP-1::GFP alone, but less than either N2 worms or the tdp-1(ok803) deletion mutant  mutant larvae show near complete dauer formation when grown at 25°C, but this phenotype was not altered in daf-2(e1370);tdp-1(ok803) mutants  worms were completely resistant  mutants were more sensitive to juglone than N2 worms, and tdp-1(ok803) completely abolished daf-2's resistance to juglone in the daf-2(e1370);tdp-1(ok803) double mutant strain  animals were more sensitive than wild type N2 worms, and tdp-1(ok803) sensitized the normally resistant daf-2 animals to stress-induced mortality (tdp-1 is required for daf-2 mediated resistance to oxidative stress. Given the effect of tdp-1(ok803) on daf-2 mutants' stress resistance compared to the lack of effect on dauer phenotypes we wondered if tdp-1 might have a more specific role in the stress response axis of IIS.To further define the role of esistant . If tdp-t strain . To corrortality . These dtdp-1 mutants were sensitive to hypertonic stress from NaCl (daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were resistant to this hypertonic stress suggesting that tdp-1 may not function, or may function in parallel to the IIS pathway to regulate the response to osmotic stress. To confirm this hypothesis we used sorbitol, another hypertonic stress producer, and similarly observed that tdp-1(ok803) mutants were sensitive to osmotic stress but daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were both resistant after either 14 hours  worms, as well as daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants in response to these stresses  animals had a modest increase in lifespan, they were statistically indistinguishable from wild type N2 worms in stress assays  or sorbitol, increased temperature, low oxygen and ultraviolet irradiation. Compared to N2 worms, rom NaCl . Interes14 hours  or 48 ho14 hours . Finallystresses –S2E. Thus assays .tdp-1. We used RT-PCR to examine expression levels of three DAF-16 genes including the catalases ctl-1 and ctl-2, as well as the superoxide dismutase sod-3 in various genetic backgrounds. First of all, no expression of tdp-1 was observed in strains containing the deletion allele tdp-1(ok803)  (sod-3 expression was greatly reduced in daf-2(e1370);tdp-1(ok803) animals  . Next we2(e1370) . However animals . These dtdp-1(ok803) mutants to oxidative and osmotic stresses we next wanted to see if there were in vivo changes in TDP-1 expression in response to these two stress conditions. In wild type unstressed animals TDP-1::GFP is lowly expressed (tdp-1 was not required for expression of the TDP-1::GFP transgene since expression was induced by stress in tdp-1(ok803) deletion mutants ;TDP-1::GFP strain (daf-2(e1370) mutants exposed to stress  mutation ;TDP-1::GFP transgenics to stress. Exposure to NaCl continued to induce TDP-1::GFP expression in daf-16 mutants  mutants was abolished by daf-16(RNAi) directly linking the IIS pathway to tdp-1 expression (tdp-1(ok803) had no effect on DAF-16::GFP stress-induced nuclear localization  mutation TDP-1::GFP was no longer restricted to the nucleus and was observed in the cytoplasm  and daf-2(e1368) mutants under normal and stressed conditions. As seen with our TDP-1::GFP reporter strain, we observed that TDP-1 protein levels were elevated in daf-2(e1370) animals, and we observed a further increase in TDP-1 levels in daf-2(e1370) animals exposed to oxidative stress (daf-2(e1368);TDP-1::GFP animals, we observed that TDP-1 protein levels remained low in daf-2(e1368) animals under normal and stress conditions ;TDP-1::GFP reporter strain. In summary, TDP-1 is a stress responsive protein whose expression is greatly influenced by the IIS pathway especially in the context of oxidative stress.To make certain that the induction of TDP-1 by stress was not a phenomenon restricted to our transgenic reporter strain we examined endogenous TDP-1 protein levels under normal and stress conditions. Using a monoclonal antibody against ns TDP-1  and westns TDP-1 . To confe stress . Consistnditions . Finally juglone  in agreeOur findings begin to outline a complex role for TDP-1 in lifespan and the cellular stress response in relation to the IIS pathway. As one of our main interests is understanding the role of aging and stress signaling in the context of age-dependent neurodegeneration we next investigated how proteotoxicity contributed to these processes.C. elegans Hsp70 protein orthologous to mammalian Grp78/BiP, and the transgenic C. elegans hsp-4p::GFP reporter is activated in response to misfolded proteins within the endoplasmic reticulum (ER), including chemically by compounds like tunicamycin (hsp-4p::GFP expression (ER). We did not observe induction of GFP in reporter strains for the mitochondrial chaperones hsp-6/Hsp70 and hsp-60/Hsp10/60 or the cytoplasmic chaperone hsp-16.2/Hsp16 , a compound that fluoresces when exposed to intracellular peroxides associated with oxidative stress ER mutant TDP-43 and FUS generate oxidative stress. To further establish the link between proteotoxicity and the IIS we examined the subcellular localization of DAF-16. DAF-16::GFP is typically cytoplasmic under non-stressed and/or low insulin signaling conditions (A consequence of processing misfolded proteins within the ER is the production of reactive oxygen species as part of what is termed the integrated stress response nditions . Consistnditions .daf-2 function suppresses proteotoxicity daf-2 mutations have opposite effects on TDP-1 expression and if elevated TDP-1 expression were cytotoxic then we would expect to see opposing effects of daf-2(e1368) and daf-2(e1370) on TDP-43 toxicity in our models. To examine this directly we created daf-2(e1368);TDP-43[A315T] and daf-2(e1370);TDP-43[A315T] strains and scored paralysis phenotypes. Interestingly, we observed that daf-2(e1368) suppressed paralysis while daf-2(e1370) enhanced paralysis compared to TDP-43[A315T] alone  significantly reduced the motor neuron degeneration caused by mutant TDP-43 daf-2(e1370) enhanced degeneration  and pellet (detergent-insoluble) and by western blotting with human TDP-43 antibodies we have previously shown that mutant TDP-43 is prone to aggregation and is highly insoluble daf-2(e1368);TDP-43[A315T], and daf-2(e1370);TDP-43[A315T] animals we observed that daf-2(1368) greatly reduced the amount of insoluble TDP-43 compared to control transgenics while daf-2(e1370) had no effect  and FUS[S57Δ];tdp-1(ok803) double mutant strains and observed that these animals had a significantly lower rate of paralysis compared to single transgenic TDP-43[A315T] or FUS[S57Δ] worms (tdp-1(ok803) mutants (tdp-1(ok803) strains co-expressing mutant TDP-43 or mutant FUS, we examined the solubility of mutant TDP-43 and FUS proteins with our biochemical assay tdp-1 suggesting that the protective effects are not due to down-regulation or clearance of mutant proteins  mutants are truly linked to this gene based on several lines of experimental evidence.Although the n Signal  and any tdp-1(RNAi) reduces the long-lived phenotype of daf-2(e1370) worms in agreement with the tdp-1(ok803);daf-2(e1370) strain mutant. We also tested if introducing wild type tdp-1 DNA sequence could rescue the long-lived phenotype of tdp-1(ok803) mutants. Using a strain expressing a full length TDP-1 open reading frame driven by the endogenous tdp-1 promoter we observed that it could partially rescue the extended lifespan phenotype of tdp-1(ok803) mutants. This is a direct proof that the lifespan phenotypes we observe in tdp-1(ok803) mutants is due to loss of the sequence and that we can correct this phenotype by introduction of wild type tdp-1 sequence.Concerning the lifespan phenotypes, tdp-1 in the cellular stress response with several experimental approaches. First, tdp-1(ok803) mutants are sensitive to oxidative and osmotic stress and we observed that our TDP-1::GFP reporter is induced by these same stresses. Second, western blotting with a TDP-1 antibody showed that endogenous TDP-1 protein levels are also induced by oxidative and osmotic stress. Third, our genetic experiments with tdp-1(ok803) showed that these mutants are sensitive to oxidative stress via the IIS pathway, but sensitivity to osmotic stress is independent of the IIS pathway. Our experiments with the TDP-1::GFP reporter or immunoblotting with the TDP-1 antibody fully support the observations made with the tdp-1(ok803) mutant. Finally, tdp-1(ok803) suppresses proteotoxicity while TDP-1::GFP enhances toxicity. Again, this is akin to a classic genetic rescue experiment and provides direct evidence that the phenotype observed by deletion of tdp-1 can be modified by the introduction of wild type tdp-1 sequence. In total we believe our hypothesis that tdp-1 has roles in lifespan and stress is well supported by multiple approaches. It is not clear why we see very little effect for the ok781 allele in stress assays, but at the same time there is not yet general consensus for any of the phenotypes observed for the tdp-1 mutants.We corroborated the role of ER to restore homeostasis ER are protective but ER stress also stimulates the clearance of misfolded proteins from the ER through ER-associated degradation (ERAD) by transporting misfolded proteins from the ER lumen to the cytoplasm for degradation by the ubiquitin-proteasome. ERAD is energetically costly, redox intense and leads to substantial production of oxidative stress and if the ER stress is not resolved it can lead to cell death An early location for mutant TDP-43 and FUS toxicity may be within the ER. The ER has critical cellular functions including protein folding, and misfolded proteins within the ER cause stress leading to activation of the UPROur model complements the “two-hits” hypothesis for sporadic diseases that highlights the role of environmental stresses in combination with cytoplasmic accumulations of TDP-43 as part of the trigger for pathogenesis C. elegans by creating transgenic strains that either overexpressed stress-activated TDP-1 or were missing the worm's endogenous tdp-1. Paralysis and lifespan phenotypes of TDP-43 and FUS transgenics were worsened by increased expression of TDP-1. Consistently, deletion of endogenous tdp-1 from strains expressing mutant TDP-43 or FUS remarkably reduced paralysis and motor neuron degeneration phenotypes. Taken together we directly showed that wild type tdp-1 plays an important role in neurodegeneration caused by mutant TDP-43 and FUS proteins.Many TDP-43 models have been described and they all show toxicity from the over expression of wild type TDP-43, which is sometimes less toxic than mutant but not always C. elegans have shown no effect on TDP-43 toxicity in animals mutant for endogenous tdp-1tdp-1 reduces TDP-43 and SOD1 toxicity tdp-1. Additionally, the fact that we see a reduction of mutant FUS toxicity by deleting tdp-1 bolsters the notion that proteotoxic induction of TDP-1 propagates toxicity and this may not be a phenomenon restricted to mutant TDP-43.However two studies in ER appears to be activated in a cell non-autonomous manner. The hsp-4p::GFP reporter is expressed primarily in the worms intestinal cells while mutant TDP-43 and FUS are expressed in motor neurons. Thus ER stress generated within neurons is capable of signaling to other cells and tissue types perhaps as part of a coordinated organism wide response. Cell non-autonomous signaling has been described in C. elegans for mitochondrial stress regulating longevity A surprising finding is that the UPRtdp-1 and daf-2. tdp-1(ok803) mutants have extended lifespan but are stress sensitive, while daf-2(e1370);tdp-1(ok803) mutants are stress sensitive and have decreased lifespan compared to daf-2(e1370) alone. In all systems examined increased TDP-43 expression is toxic tdp-1 from worms leaves them sensitive to stress but frees them from potential long-term cytotoxic effects. We are not alone in the observation that removing tdp-1 increases lifespan tdp-1's role in the cellular stress response. Looking at daf-2, the difference here may be that tdp-1 is essential for the stress resistance aspects of daf-2 mutants and concomitant long-lived phenotype. Thus removing tdp-1 renders daf-2 animals sensitive to stress induced damage limiting their extended lifespan.Concerning the regulation of lifespan, our work describes a complex relationship between daf-2 mutations on proteotoxicity and TDP-1 expression. Several publications have reported that reduced daf-2 function suppresses proteotoxicity daf-2(RNAi) or on a single daf-2 allele (e1370) to investigate proteotoxicity while we looked at two different daf-2 alleles, e1368 and e1370. Further complicating the matter is the fact that some models are based on muscle-cell expression vectors and/or have severe developmental effects like the recently described TDP-43 model A surprising finding was the opposing effects of daf-2 mutations are grouped into a complex allelic series comprising two classes. Class 1 alleles are less severe and mutations fall within the extracellular regions of the DAF-2 receptor protein daf-2(e1368) and the class 2 allele daf-2(e1370) we observed that the two alleles had opposing effects on mutant TDP-43 toxicity and TDP-1 expression. In our mutant TDP-43 worms we observed that daf-2(e1368) suppressed mutant TDP-43 induced paralysis, while the class 2 allele daf-2(e1370) enhanced paralysis. Furthermore, daf-2(e1368) reduced the insolubility of mutant TDP-43 protein, while daf-2(e1370) had no effect on mutant TDP-43 solubility. Using a TDP-1::GFP reporter or western blotting with a TDP-1 antibody we observed that daf-2(e1370) increased the expression of TDP-1 while daf-2(e1368) had no effect. Given that the IIS pathway regulates both the toxicity of mutant TDP-43 proteins and the expression of endogenous TDP-1/TDP-43, altered IIS may directly contribute to ALS pathogenesis.daf-2 are known and they are consistent within each class daf-2(e1368) mutants are sensitive to hypoxia while class 2 daf-2(e1370) mutants are highly resistant to hypoxia daf-2 on proteotoxicity and the cytotoxic induction of TDP-1 expression both of which are consistent for each allele. Our data are in agreement with studies showing that reduced IIS diminishes proteotoxicity but the question remains as to why the more severe class 2 daf-2(e1370) allele enhances toxicity in our TDP-43 transgenics. Part of the reason may lie with intrinsic differences between models. Our transgenics are based on the expression of TDP-43 in 26 GABAergic neurons and the worms do not show motor impairment until well into adulthood daf-2 mutations are required to suppress severe phenotypes but are too strong for the milder, late-onset toxicity in our TDP-43 transgenics. There may be an optimal rate of IIS unique to each situation that has been referred to the Insulin Signalling Paradox The pleiotropic and sometimes opposite effects of ER stress by promoting protein folding. In the future it will be important to determine if strategies to reduce TDP-43 neuronal toxicity may be applicable to additional neurodegenerative disease as a shared mechanism of cell death in the development of new therapeutics.It is still unclear if mutant TDP-43 results in a gain, a loss of function or both but work from zebrafish and fly TDP-43 models suggest that it may be both Caenorhabditis elegans Genetics Center CGC . All strains were maintained following standard methods on OP50 bacteria plates. Strains used in this study include: tdp-1(ok803) and tdp-1(ok781) both outcrossed 5 times to N2 prior to use, daf-2(e1368), daf- 2(e1370), daf-16(mu86), gpIs1[hsp-16.2::GFP], oxIs12, xqIs93[tdp-1p::TDP-1::GFP], xqIs132, xqIs133[unc-47p::TDP-43[A315T];unc-119(+)], xqIs173, xqIs98[unc-47p::FUS[S57Δ];unc-119(+)], zIs356, zcIs4[hsp-4p::GFP], zcIs9[hsp-60p::GFP],gpIs1[hsp-16.2p::GFP] and zcIs13[hsp-6p::GFP].Nematode strains used were described previously Sixty synchronized L4 were grown on OP50 bacteria plates  and three independent assays were performed. Lifespan analyses were performed at 20°C and 25°C and worms were scored every 1–2 days from adult day 1 until death. Worms were scored dead if they didn't respond to tactile or heat stimulus.E. coli (HT115) containing an Empty Vector (EV), daf-16 (R13H8.1), or tdp-1 (F44G4.4) RNAi clones from the ORFeome RNAi library (Open Biosystems). RNAi experiments were performed at 20°C. Worms were grown on NGM enriched with 1 mM Isopropyl-b-D-thiogalactopyranoside (IPTG). For lifespan, worms were transferred to RNAi 5-fluorodexyuridine  plates at adult day 1 until death. Worms were declared dead if they didn't respond to tactile or heat stimulus. Experiments were conducted with 20 animals/plate by triplicates.RNAi-treated strains were fed with daf-2(e1370) were allowed to lay eggs overnight at 20°C. The eggs were then transferred to 25°C and scored for dauer formation 5 days later. Three different trials on different days were performed.Young adult Stress tests were performed at 20°C , 25°C (hypoxia) and 37°C . Worms were grown on NGM and transferred to NGM plates + 240 µM juglone (oxidative stress), or NGM plates + 10 mM Hydrogen peroxide (oxidative stress), or NGM plates + 400 mM NaCl (osmotic stress), or NGM plates + 611 mM Sorbitol (osmotic stress), all at adult day 1. For the oxidative, osmotic and thermal stress assays, worms were evaluated for survival every 30 minutes for the first 2 hours and every 2 hours after up to 14 hours; for sorbitol we also performed a test over 48 hours, starting the counts after 14 hours on the compound. For UV irradiation, adult day 1 worms were transferred to NGM plates without any food source and exposed to UV (1200 J/m2). Worms were then transferred to NGM plates with OP50 bacteria and died animals were counted every 2 hours till 14 hours after irradiation. For hypoxia experiments young adult animals were transferred to a new plate and subjected to low oxygen conditions with the AnaeroPack system  for 24 hours and assayed for survival. For all experiments nematodes were scored as dead if they were unable to move in response to heat or tactile stimuli. For all tests worms, 20 animals/plate by triplicates were scored.tdp-1 promoter and open reading frame plasmid clones were obtained from Open Biosystems and recombined with plasmid pDES-MB14 , and verified by sequencing to create a tdp-1p::TDP-1::GFP plasmid, which was injected at 5 ng/µl into unc-119(ed3) animals along with myo-3p::mCherry, myo-2p::mCherry comarkers at 5 ng/µl and wild type transformants expressing GFP were kept. The transgene was integrated using UV radiation and wild type, GFP positive animals were kept for further study. Multiple stable transgenics were isolated and outcrossed to N2 4 times before use. Strain XQ93 xqIs93[tdp-1p::TDP-1::GFP] was used in this study.Gateway system (Invitrogen) compatible TDP-1::GFP animals, M9 buffer with 5 mM levamisole was used for immobilization. Animals were mounted on slides with 2% agarose pads. TDP-1::GFP expression was visualized with a Leica CTR 6000 and a Leica DFC 480 camera. L4 animals were grown on NGM plates and transferred to NGM plates + 240 µM juglone (oxidative stress) or NGM plates + 400 mM NaCl (osmotic stress) for 90 minutes, and examined for fluorescence with the Leica system described above. Some animals were also stained with DAPI . Image processing was done with Adobe Photoshop. For images of TDP-1::GFP alone, images were converted to black and white and the images reversed to allow for better contrast and visualization. Quantification of TDP-1::GFP levels was done with ImageJ (NIH) and the mean and SD was calculated from 5 images for each strain and experimental condition. For visualization of DAF-16::GFP, hsp-4p::GFP, hsp-6p::GFP, hsp-16.2p::GFP and hsp-60p::GFP animals, M9 buffer with 5 mM levamisole was used for immobilization. Animals were mounted on slides with 2% agarose pads and examined for fluorescence with the Leica system described above.For visualization of For visualization of oxidative damage in the transgenic strains the worms were incubated on a slide for 30 minutes with 5 mM dihydrofluorescein diacetate dye (Sigma-Aldrich) and then washed with 1× PBS three times. After the slide was fixed fluorescence was observed with the Leica system described above.ctl-1 forward, AGGTCACCCATGACATCACCAAGT; ctl-1 reverse, GAT TGCGCTTCAGGGCATGAATGA; ctl-2 forward, TTCGCTGAGTTGAACAATCCG; ctl-2 reverse, GTTGCTGATTGTCATAAGCCATTGC; tdp-1 forward, AAAGTGGGATCGAGTGACGAC; tdp-1 reverse, GACAGCGTAACGAATGCAAAGC; sod-3 forward, CGAGCTCGAACCTGTAATCAGCCATG; sod-3 reverse, GGGGTACCGCTGATATTCTTCCACTTG; act-3 forward, GTTGCCGCTCTTGTTGTAGAC; act-3 reverse, GGAGAGGACAGCTTGGATGG.RNA was extracted with an RNAeasy kit (Qiagen) and reverse transcribed with QuantiTect (Qiagen). Primers used include: 1/2 G syringe 10 times, sonicated and centrifuged at 16000 g. Supernatants were collected.Worms were collected in M9 buffer, washed 3 times with M9 and pellets were placed at −80°C overnight. Pellets were lysed in RIPA buffer  + 0.1% protease inhibitors . Pellets were passed through a 271/2 G syringe 10 times, sonicated and centrifuged at 100000 g for 5 minutes. The soluble supernatant was saved and the remaining pellet was resuspended in extraction buffer, sonicated and centrifuged at 100000 g for 5 minutes. The remaining pellet was resuspended into 100 µl of RIPA buffer, sonicated until the pellet was resuspended in solution and saved.For TDP-43 and FUS transgenics, soluble/insoluble fractions, worms were lysed in Extraction Buffer . Pellets were passed through a 27All supernatants were quantified with the BCA Protein Assay Kit (Thermo Scientific) following the manufacturer instructions.Worm RIPA samples  were resuspended directly in 1× Laemmli sample buffer, migrated in 10% polyacrylamide gels, transferred to nitrocellulose membranes (BioRad) and immunoblotted. Antibodies used: rabbit anti-TDP-43 , rabbit anti-FUS/TLS , rabbit anti-TDP-1  and mouse anti-Actin . Blots were visualized with peroxidase-conjugated secondary antibodies and ECL Western Blotting Substrate (Thermo Scientific). Densitometry was performed with Photoshop (Adobe).t-tests were used for statistical analysis.For lifespan and stress-resistance tests, survival curves were generated and compared using the Log-rank (Mantel-Cox) test, and 20–30 animals were tested per genotype and repeated at least three times. For progeny counts, dauer-formation assays and hypoxia tests the mean and SEM were calculated for each trial and two-tailed Figure S1C. elegans TDP-1. A) Comparison of human TDP-43 and C. elegans TDP-1 proteins. The extent of the deletion allele ok803 is indicated. RRM: RNA Recognition Motif, NLS: Nuclear Localization Signal. (B) Detection of the deletion alleles tdp-1(ok781) and tdp-1(ok803) by PCR with oligonucleotides spanning both deletions. Non-mutant N2 worms produce a band of ∼1600 bp, tdp-1(ok803) an ∼430 bp band, and tdp-1(ok781) a ∼480 bp band.Human TDP-43 and (TIF)Click here for additional data file.Figure S2tdp-1 is not required for dauer formation or resistance to heat, hypoxia or radiation. (A) tdp-1(ok803) did not interfere with the constitutive dauer-formation phenotype of daf-2(e1370) animals grown at 25°C. (B) tdp-1(ok803) worms were more sensitive to osmotic stress from sorbitol than wild type N2 worms (P<0.001). daf-2(e1370) and daf-2(e1370);tdp-1(ok803) were statistically indistinguishable from one another in their response to sorbitol but were both highly resistant compared to N2 controls (P<0.001). (C) tdp-1(ok803) mutants and N2 worms showed similar sensitivity to thermal stress. daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were highly resistant to thermal stress compared to N2 controls (P<0.001). (D) Wild type N2 worms and tdp-1(ok803) mutants were both highly susceptible to mortality caused by low oxygen conditions. tdp-1(ok803) did not interfere with the resistance of daf-2(e1370) animals against hypoxia. (E) tdp-1(ok803) mutants and N2 worms were equally sensitive to UV radiation. daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were both highly resistant to UV compared to N2 controls (P<0.001).(TIF)Click here for additional data file.Figure S3tdp-1(ok781) has variable phenotypes. (A) tdp-1(ok781) mutants show a modest but significant increase in lifespan compared to N2 worms. tdp-1(ok781) worms were indistinguishable from N2 worms in their response to (B) juglone induced oxidative stress, (C) NaCl induced osmotic stress and (D) thermal stress. Please also see (TIF)Click here for additional data file.Figure S4tdp-1(ok803);TDP-1::GFP in untreated and animals exposed to either NaCl or juglone. *P<0.001 compared to untreated animals. Linked to daf-2(e1370);TDP-1::GFP and daf-2(e1368);TDP-1::GFP animals under normal or stressed conditions. *P<0.001 compared to untreated animals. Linked to daf-16(mu86);TDP-1::GFP animals under normal and stress conditions. *P<0.001 compared to untreated animals. Linked to daf-2(e1370);TDP-1::GFP animals exposed to daf-16(RNAi) or empty vector controls. *P<0.001 compared to untreated animals. Linked to Quantification of TDP-1::GFP expression. (A) Quantification of TDP-1::GFP expression in transgenics exposed to osmotic stress (NaCl) or juglone oxidative stress (juglone). *P<0.001 compared to untreated animals. Linked to (TIF)Click here for additional data file.Figure S5unc-47p::GFP compared to untreated controls. (B) sod-3p::GFP expression was induced by oxidative or osmotic stress compared to untreated controls.Stress assays do not induce general expression of transgenes. (A) Oxidative or osmotic stress did not induce the expression of (TIF)Click here for additional data file.Figure S6daf-2(e1370) mutants and was unaffected by empty vector RNAi controls. (B) daf-16(RNAi) abolished the increased expression of TDP-1::GFP in daf-2(e1370) mutants. (C) DAF-16::GFP is cytoplasmic and diffuse under normal conditions but is (D) localized to nuclei when the animals are exposed to juglone. (E) The GFP signal is diffuse in unstressed daf-16::GFP;tdp-1(ok803) transgenics and (E) localizes within the nuclei of transgenics treated with juglone.TDP-1 functions downstream of DAF-16. (A) TDP-1::GFP expression was upregulated in (TIF)Click here for additional data file.Figure S7tdp-1(ok803) and tdp-1(ok781) strains with a polyclonal anti TDP-1 antibody revealed a signal in N2 worms but no signals from the deletion mutants ok803 or ok781.Characterization of the TDP-1 antibody. Western blotting of protein extracts from wild type N2, (TIF)Click here for additional data file.Figure S8hsp-6p::GFP under non-stress conditions. (B) Ethidium bromide induced hsp-6p::GFP expression. (C) Mutant TDP-43 did not induce hsp-6p::GFP expression. (D) Low expression of the mitochondrial chaperone reporter hsp-60p::GFP under non-stress conditions. (E) Ethidium bromide induced hsp-60p::GFP expression. (F) Mutant TDP-43 did not induce hsp-60p::GFP expression. (G) Low expression of the heat shock reporter hsp-16.2p::GFP under non-stress conditions. (H) High temperatures induced hsp-16.2p::GFP expression. (I) Mutant TDP-43 did not induce hsp-16.2p::GFP expression.Mutant TDP-43 does not activate mitochondrial or cytoplasmic chaperones. (A–I) are low-resolution photographs of young adult transgenic worms. The images have been converted to black and white and photo-reversed to aid visualization. Difficult to see worms are outlined. (A) Low expression of the mitochondrial chaperone reporter (TIF)Click here for additional data file.Table S1Lifespan analysis for all experiments. Related to (PDF)Click here for additional data file.Table S2Stress assays. Related to (PDF)Click here for additional data file.Table S3Paralysis tests. Related to (PDF)Click here for additional data file.Table S4tdp-1(ok781). Related to Lifespan and stress assays for (PDF)Click here for additional data file.


Dorsal phase of DTC migration is regulated by integrin mediated signaling. We find that ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the α-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, we investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, we propose that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement.Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of  The metastasis suppressor function has been extensively corroborated using metastatic cell lines  where, for the most part, overexpression of NM23-H1 was associated with reduced cell motility (reviewed in nm23 family are classified into two groups. Isoforms of group I (NM23-H1–NM23-H4) possess nucleoside diphosphate kinase activity and are highly conserved in eukaryotes from yeast to mammals The human Drosophila. awd, the fly orthologue of nm23-H1/H2 is a negative regulator of migrating tracheal and border cells via modulating endocytosis of different receptors, such as platelet-derived growth factor receptor (PDGFR)/vascular endothelial growth factor receptor (VEGFR) Drosophila, in rat a direct interaction was detected between NDPK and dynamin I by in vitro pulldown and coimmunoprecipitation Dictyostelium also confirm links to light-dependent, vectorial cell migration and cell nutrition through different forms of endocytosis The most extensive set of studies analyzing group I members' role in cell motility and migration have utilized Caenorhabditis elegans serves as a particularly amenable model to investigate the process of cell migration. The nematodes are transparent and have simple anatomy making it possible to follow the migration of individual cells in the living animal throughout development. Well studied migrating cell types of C. elegans include sex myoblasts (SM), two Q neuroblasts (QL and QR) and their descendants, and distal tip cells (DTCs) or the gonadal leader cells C. elegans, we identified and described a single group I NDPK ortholog, NDK-1, which shows 85% and 86% similarity to NM23-H1 and H2, respectively C. elegansThe tightly regulated migratory path of DTCs provides an elegant system to explore how cell migration can be guided within the spatial and temporal context of the organism. Distal tip cells are specialized leader cells and are responsible for gonad morphogenesis via their migration in C. elegans /Rac and in parallel to abi-1 (Abl interactor)/ABI; and additionally shows a genetic interaction with dyn-1/dynamin. Thus, NDK-1 affects the rearrangement of cytoskeleton in both DTC migration and apoptotic engulfment. We also show that NDK-1 functions similar to its human counterparts in cell migration, as it inhibits the migratory potential of invasive breast adenocarcinoma cells. Our results might help to better understand the function of nm23 genes in metastasis.In this study we demonstrate that NDK-1 is required for normal DTC migration and engulfment of apoptotic corpses. We show that C. elegans. NDK-1 is the single group I NDPK homolog of the worm and shows high sequence similarity to NM23-H1 and H2 nm23 genes regulate cell migration C. elegans homolog of NM23-H1/H2 is also able to act likewise. Therefore we investigated the effect of NDK-1 exerted on the cell migration capacity of the breast adenocarcinoma MDA-MB-231T cell line. MDA-MB-231T cells are far more migratory than CAL27 cells, and the influence of NM23-H1 is much more obvious in these cells. Stably transfected MDA-MB-231T cells overexpressing FLAG::NDK-1, FLAG::NM23-H1 and MYC-NM23-H2 (Our group is investigating the function of nucleoside diphosphate kinases (NDPKs) in the model organism ndk-1, and identified the strong loss of function allele ok314. ok314 is a 1157 bp-long deletion, which removes the entire ndk-1 ORF, as well as upstream and downstream regulatory sequences We recently characterized the worm ortholog of group I NDPKs, ndk-1(ok314) mutants show a Pvl (protruding vulva) phenotype and they are sterile due to germ cell arrest in mitotic phase ndk-1(ok314) animals have insufficiently elongated gonad arms suggesting defects in DTC migration. Detailed analysis (ok314 mutants (60.3%) DTCs turned to the proper direction and side however their migration is stopped prior to reaching the vulva. In 14.5% of the cases DTCs turned back to the vulva but in the ventral  side or initiated ventral migration but subsequently vectored to the dorsal side before the turn (7.2%). Other defects manifested low penetrance  and we observed normal DTC migration in only 4.7% of the animals. Altogether we conclude that the prominent phenotype of ok314 mutants is incomplete migration of DTCs.In order to determine whether NDK-1 is expressed in DTCs, we studied NDK-1 expression in transgenic strains TTV2 and TTV3. Both were generated using the same translational construct but by distinct methods. In case of TTV2, the strain was created by ballistic bombardment, which resulted in extrachromosomal arrays of the transgene and therefore genetic mosaicism. For TTV3 the MosSCI method was applied to integrate a single copy of the transgene into the genome. The latter method allowed the rescue of Ndk-1 mutant phenotypes. Among TTV2 and TTV3 animals we observed expression in DTCs from L3  and one β (pat-3) subunits C. elegans integrins are essential, null mutations in any of them cause embryonic lethality ina-1 mutants show an abnormal migration  phenotype because DTCs do not stop prior to reproduction pat-2 by RNAi resulted in dorsal pathfinding defects pat-3 transgene or by pat-3(RNAi) The vab-3  encodes a homeodomain protein, the C. elegans orthologue of Pax6. vab-3/Pax6 transcriptionally regulates both α-integrins in different manners: it downregulates ina-1  mutants.migration) have been shown to act downstream of INA-1 during the migration of DTCs ced-10 was found to function together with ced-2 and ced-5 in DTC migration  and abl-1(n1963) alleles manifest no obvious gonadal phenotype ced-2, ced-5, ced-12 and ced-10 mutants were suppressed in abl-1 mutant backgrounds. ABL-1 inhibits ABI-1 in C. elegans, abi-1(tm494) mutation itself or abi-1(RNAi) caused only weak defects in DTC migration abi-1(RNAi) enhanced the extra turns of ced-5(n1812) and ced-12(n3261) abi-1(ok640) mutants and observed 26% of abnormal gonad arms in which the overshoot phenotype was the most frequent (13%) , ina-1(gm39), ced-10(n1993), mig-2(lq13), unc-73(e936), ced-5(n1812), ced-12(n3261), abl-1(ok171)/ABL1, abi-1(ok640). Except abl-1(ok171) and abi-1(ok640), all these mutants display large gonads with extra turns  using the vab-3(e1796), ina-1(gm39), unc-73(e936), ced-5(n1812) and ced-12(n3261) RNAi was applied to knockdown NDK-1 function in the corresponding mutant background. In the cases of mig-2(lq13), ced-10(n1993), abl-1(ok171) and abi-1(ok640) double mutants were created by introducing the ok314 mutant allele of ndk-1 into the appropriate single mutants.To examine the effect of the absence of NDK-1 in the above mentioned mutant backgrounds, we either depleted the function of NDK-1 by RNAi or generated double mutants. In the cases of ndk-1 suppressed the extra turns of vab-3, ina-1, unc-73, mig-2, ced-5, ced-12 and ced-10 mutants and mostly resulted in Ndk-1-like gonad (reduced gonad with fewer extra turns) (abl-1(ok171) was able to suppress the phenotype of the ced mutants ndk-1 mutants. Unlike abl-1(ok171), abi-1(ok640) mutation was able to restore the DTC migration defects of ndk-1(ok314) animals in 37% of the cases  loss of function mutants arrest at the mitotic phase before reaching pachytene (our unpublished data), thus engulfment defects cannot be investigated in this mutant background. We analyzed the gonads of ndk-1(RNAi) worms using DIC optics and also marked the early apoptotic corpses with CED-1::GFP ndk-1(RNAi) treated worms  compared to the control animals   and in wild type embryos around the comma stage. We observed a near doubling of corpses in ndk-1 mutant embryos (20.1) compared to wild type (10.9), indicating that NDK-1 plays a role in apoptosis .During ndk-1 and the key positioned genes abi-1 and ced-10 . First we counted apoptotic corpses in the homozygous mutant progeny of ndk-1(ok314)/+;ced-10(n1993) animals and found that double mutant comma stage embryos contained on average 25.2 apoptotic corpses  which is comparable with ced-10(n1993) single mutants; 24.8. The majority of the 25 examined ndk-1(ok314);ced-10(n1993) double mutant embryos displayed cell corpses between 22–27, reminiscent of ced-10(-) single mutants, however around 10% of the doubles showed an increased number of cell corpses (e. g. in two embryos 38 corpses were counted)  harboring mutations in abi-1(tm494) or abi-1(RNAi) alone caused only weak defects in engulfment abi-1(ok640) embryos (18.7) compared to wild type (10.9) /+;abi-1(ok640) heterozygotes revealed that ndk-1(ok314);abi-1(ok640) double mutants displayed 36.7 cell corpses on average . Thus, introducing abi-1(ok640) mutation into ndk-1(ok314) background we noticed an additive phenotype, an increased level of apoptotic corpses  should not be more severe than that of the stronger single (null) mutant. If the two genes function in parallel pathways a (null) mutation in one of them should enhance significantly the phenotype caused by the other, resulted in an additive phenotype ndk-1 acts in parallel to abi-1 and downstream of ced-10 /+ heterozygotes 12% of ndk-1(ok314) homozygotes die as embryos ndk-1(ok314) embryos also show the Dyn-1-like late embryonic lethality phenotype with persistent cell corpses ;dyn-1(-) double mutants. For crossing we used ndk-1(ok314) mutants and the viable ky51 allele of dyn-1, wherein we took the advantage of thermosensitivity.Next, to see whether ky51 (25°C) (ndk-1(ok314) homozygotes died as embryos and ndk-1/+ heterozygotes laid on the average 122.7 eggs. We measured approximately 50% lethality of dyn-1(ky51) single mutants and 51% lethality in the F1 progeny of dyn-1(ky51);ndk-1(ok314)/+ animals, but the broodsize was decreased (43.9) in the latter category compared to dyn-1 single mutants (63). At the restrictive temperature we did not notice any Pvl, Ste (double mutants) in the F1 progeny. Putting the F1 animals to the permissive temperature (15°C) the Pvl, Ste phenotype appears in F2 generation. Therefore these observations suggest that the double mutant is not viable indicating that ndk-1 and dyn-1 interact genetically in the worm as well.We performed this experiment at the restrictive temperature for C. elegans serves as a tractable model system to monitor the effects of the NDPK gene family.Group I NDPKs are negative regulators of cell migration and motility. To investigate the function of the sole worm group I homolog, NDK-1 in cell migration, we overexpressed NDK-1 and human NM23-H1 and H2 in MDA-MB231T, an invasive breast carcinoma cell line. We found that NDK-1, similar to its human counterparts, dramatically suppressed the migratory potential of MDA-MB231T cells. This result shows that NDK-1 bears an evolutionary conserved function in cell migration and that ndk-1 in the process of the migration of distal tip cells (DTCs) which are responsible for leading gonad morphogenesis. During larval development distal tip cells guide the migrating gonad arms which finally form two U-shaped tubes ndk-1 loss of function mutants showed different DTC migration defects, but predominantly, incompletely elongated gonad arms were the consistent feature. The majority of ok314 mutants showed a third  phase migratory defect, as a consequence of a premature stop of DTCs, after the turn, that was not present in wild-type worms.Next, we studied the role of Dictyostelium growth in opposing ways in different cellular environment Thus, overexpression of NDK-1 inhibited cell migration and reduced motility of metastatic breast adenocarcinoma cells, but loss of NDK-1 function in the worm predominantly caused incomplete gonadal migration and not excess migration. These data suggest that NDK-1 might have opposing functions depending on the cellular environment, e.g. NDK-1 might promote cell migration in the worm, but inhibit it in human cell lines. In support of this idea, recent work showed that NDPK affects nm23 genes were linked to integrin signaling in human studies C. elegans, integrins act in the dorsal/third phase of DTC migration ndk-1(ok314) mutants are also observed. Consistently, NDK-1::GFP expression was detected in the third phase of migration (from L3 to adulthood). Based on these findings we performed an epistasis analysis with genes involved in CED-10/Rac signaling  and with abl-1/ABL and abi-1/ABI which were shown to act in parallel to the CED-10 pathway. All mutants of CED-10 pathway genes have large gonads with extra turns. Putative null mutant alleles of abl-1 (ok171 and n1963) display no obvious gonad phenotype abi-1(ok640) mutants in which the overshoot phenotype was the most frequent (14%). This result is consistent with previous data, e.g. abi-1 mutations or abi-1(RNAi) results in overextensioned DTC migration ndk-1 reduced the extra turn phenotype of vab-3, ina-1, unc-73, mig-2, ced-5, ced-12 and ced-10 mutants and mostly resulted in Ndk-1-like gonad (similar to that of ndk-1(-) mutants). These results lead to the hypothesis that NDK-1 might act downstream of or in parallel to ced-10. Although abl-1(ok171) was able to suppress the extra turn phenotype of the ced mutants ndk-1 mutants. Unlike abl-1(ok171), abi-1(ok640) mutation was able to restore partially the DTC migration defects of ndk-1(ok314) animals, showing a genetic interaction between ndk-1 and abi-1.Previously nm23 has been implicated in apoptosis C. elegans, during somatic cell death neighboring cells engulf the nascent apoptotic corpses; however germ cell corpses are eliminated by specialized engulfing cells, the sheath cells ndk-1(RNAi) animals and in ndk-1(ok314) embryos, furthermore, NDK-1 is expressed in sheath cells (this study) as well as in embryos ndk-1(-) single mutants.Both distal tip cell migration and engulfment of apoptotic cell corpses require precise regulation of cytoskeleton to extend cell surfaces ndk-1 genetically interacts with abi-1 in DTC migration and functions likely downstream of ced-10, we hypothesized that ndk-1 regulates engulfment in the same manner. We analyzed ndk-1(-), abi-1(-) and ced-10(-) single mutant embryos and ndk-1(-);abi-1(-) and ndk-1(-);ced-10(-) double mutants. ced-10(-), ndk-1(-) and abi-1(-) mutants contain more apoptotic corpses than wild type animals. Moreover, we observed an increased number of corpses, an additive phenotype in ndk-1(-);abi-1(-) double mutant embryos, whereas ndk-1(-);ced-10(-) double mutants displayed apoptotic corpses reminiscent to ced-10(-) single mutants. These results suggest that ndk-1 acts in parallel to abi-1 and downstream of ced-10 during apoptotic engulfment.Starting from the observation that ndk-1 and dyn-1/dynamin, which is a downstream factor of the CED-1 pathway involved in engulfment. The CED-1 pathway is thought to recruit membranes to extend the surface of the engulfing cell, and DYN-1 is proposed to organize vesicle transport during this process. Interaction between NDPKs and Dynamins in endocytosis is well known in other model systems ndk-1(-) and dyn-1(-) single mutants display a phenotypic similarity in the worm, the late-embryonic lethality with persistent cell corpses. Double mutant analysis revealed that ndk-1(ok314);dyn-1(ky51) animals are not viable indicating a genetic interaction between the two genes in C. elegans as well.We also analyzed the putative interaction of ndk-1 shows a genetic interaction with dyn-1/dynamin, which raises the possibility that NDK-1 might represent a converging point of the CED-1 and CED-10 pathways. Interestingly, in 10% of ndk-1(-),ced-10(-) double mutant embryos we observed an increased number of cell corpses (note that in 90% of the double mutants the corps number is similar to ced-10(-) single mutants). The convergence of the two pathways would explain this partial additivity observed in ndk-1(-);ced-10(-) double mutants. There are also data showing that silencing of dyn-1 results in a DTC migration phenotype ced-10Together, we propose that NDK-1 acts downstream of CED-10 and in parallel to ABI-1 in the process of engulfment and DTC migration. In addition, ndk-1 acts downstream of ced-10 in the regulation of engulfment and DTC migration and genetically interacts with dyn-1/dynamin. These data might contribute to our understanding of how NDK-1 exerts its function in the rearrangement of the cytoskeleton. Although NDPKs were already related to phagocytosis in different systems In summary, our genetic analysis shows that C. elegans strains were cultivated at 20°C on NGM plates seeded with OP50 bacteria TTV1 ndk-1(ok314) I/hT2[bli-4(e937) let-?(q782) qIs48]; TTV2 eluEx1;unc-119(ed3)III; TTV3 eluSi1[NDK-1::GFP+cb-unc-119(+)]III; LGI: MT11068 ced-12(n3261), CB3203 ced-1(e1735), CB936 unc-73(e936), LGIII: RB829 B0336.6(ok640) corresponds to abi-1(ok640), NG39 ina-1(gm39), LGIV: MT4434 ced-5(n1812), MT1522 ced-3(n717), MT5013 ced-10(n1993), LGV: MD701 bcIs39[P(lim-7)ced-1::GFP+lin-15(+)], LGX: CX51 dyn-1(ky51), CB3304 vab-3(e1796), LE815 mig-2(lg13), XR1 abl-1(ok171).www.wormbase.org.Information about alleles can be found at yk1105e04) was cloned into pPD129.36, and the obtained construct was transformed into HT115 (DE3) bacteria. RNAi experiments were performed at 25 °C essentially as described For RNAi through ingested dsRNAs, an XhoI digested fragment of the full-length cDNA corresponding to F25H2.5 (ndk-1(RNAi), sterile adults possessing a protruding vulva were picked. For statistical analysis DTC migration phenotypes were grouped into three classes: reduced migration, migration positioned to vulva and excess migration . As animals affected by p-values for pairwise comparisons of apoptotic cell corpse numbers were calculated using the Student's t-test.Apoptotic corpses (as refractile discs) were scored in comma stage embryos directly by microscopy BamHI and NotI sites.The insert of the construct was generated from the cDNA yk1105e04 by PCR using the following primers: 5′-cag aag atc tat gga cta caa gga cga cga cga taa gat gag caa cac tga gag aac c-3′ (which contains the flag sequence) and 5′-ata gtt tag cgg ccg ctt tat tcg tag acc cat gag ttg-3′. The insert was ligated into pCDNA3.1 using 6 cells in DMEM supplemented with 10% FBS were subjected to electroporation  with 25 μg of each of the following plasmids: pcDNA3FLAG-nm23-NDK, pcDNA3FLAG-nm23-H1, pcDNA3MYC-nm23-H2 and pcDNA3 as control. Post transfection the cells were seeded on 4×100 mm plates and incubated in DMEM supplemented with 1 mg/ml geneticin (Sigma) until development of resistant colonies. Positive clones were screened by Western blotting, propagated and stored in liquid nitrogen until further usage.MDA-MB-231T cells were transfected as follows: 4×106 cells of each clone was collected, washed in PBS and sonicated.For detection of stably transfected MDA-MB-231T clones 10The samples (10 μg) were loaded on SDS-PAGE and electrotransfered to an Imobilon-PSQ membrane (Milipore). For detection of FLAG/NDK and FLAG/H1 the anti-FLAG M2 antibody (Sigma) was used while MYC/H2 was detected using anti-MYC antibody (Santa Cruz Biotechnology). Anti-α-tubulin antibody  was used as loading control. After application of appropriate secondary antibodies the protein bands were visualized using Western Lightning Plus-ECL . The image was acquired by Alliance 4.7 (Uvitec) and assembled in Adobe Photoshop.2 at 37°C.For migration assay metastatic human breast cancer MDA-MB-231T cells were used  and allowed to settle down for 20 minutes. DMEM supplemented with 1% FBS served as a chemoattractant and was added to the lower chamber. The cells were allowed to migrate for 5 hours, after which the medium was removed, non-migratory cells were removed from upper chamber with cotton swabs while bottom of the membranes with migrated cells were washed twice in PBS, and fixed in 4% formaldehyde for 15 minutes at room temperature. The cells were stained with 0.1% crystal violet, cut out from the inserts, mounted in (DAKO) on slide, analyzed by light microscopy, and photographed. The cells from four representative images of every clone were assembled in Adobe Photoshop and counted. The experiments were performed three times.For migration assay 3×10Figure S1Monitoring apoptotic corpses in embryos slightly after the comma stage in different mutant backgrounds.A–F: Monitoring apoptotic corpses in wild-type embryos (A), ndk-1(-) (D), abi-1(-) (B), ced-10(-) (C) single mutants and ndk-1(-);abi-1(-) (E), ndk-1(-);ced-10(-) (F) double mutants using DIC optics. Embryos slightly after the comma stage were scored. Each panel shows two focal planes (A–F). Arrowheads indicate apoptotic corpses.(TIF)Click here for additional data file.


The assessment of myocardial infarction (MI) heterogeneity using late Gadolinium enhancement (LGE) MRI has been limited to static analysis at single late post-injection time. We developed a rapid single breath-hold LGE MRI method using a novel parallel imaging accelerated 3D spiral acquisition. We propose to use this technique to capture temporal variation in scar enhancement at early to late post-injection times, termed early-to-late Gadolinium enhancement (ELGE) MRI.We developed an inversion recovery 3D spiral imaging sequence with a novel iterative self-consistent parallel imaging reconstruction (SPIRiT) method, which allows entire LV coverage within only 12 heart beats. The scan parameters were: acceleration factor = 2, TI=200-300ms, spatial resolution = 1.7×1.7×7 mm3, 14 partition slices, 190 ms data acquisition window at mid-diastole, a GE 1.5 T scanner, and an 8-channel cardiac coil. Starting at 1 or 2 minutes after contrast administration, the 3D ELGE imaging was performed every minute until 10 minutes post injection. Afterwards, the standard 2D multi-slice LGE MRI was performed. The time series of 3D ELGE data were registered using a mutual information-based motion correction to compensate for different breath-hold positions.Figure Time-resolved characterization of myocardial scar tissue is feasible using 3D spiral ELGE MRI. Visual assessment and ROI-based time curve analysis demonstrated that scar enhancement is heterogeneous both spatially and temporally. Clinical validation of the proposed ELGE MRI in a large cohort of MI patients remains to be investigated.AHA postdoctoral fellowship (09POST2150025).NIH SBIR grant (5R44HL084769).


C. elegans mutations blocking the normal course of programmed cell death and clearance confer animal-wide resistance to a specific set of environmental stressors; namely, ER, heat and osmotic stress. Remarkably, this pattern of stress resistance is induced by mutations that affect cell death in different ways, including ced-3 (cell death defective) mutations, which block programmed cell death, ced-1 and ced-2 mutations, which prevent the engulfment of dying cells, and progranulin (pgrn-1) mutations, which accelerate the clearance of apoptotic cells. Stress resistance conferred by ced and pgrn-1 mutations is not additive and these mutants share altered patterns of gene expression, suggesting that they may act within the same pathway to achieve stress resistance. Together, our findings demonstrate that programmed cell death effectors influence the degree to which C. elegans tolerates environmental stress. While the mechanism is not entirely clear, it is intriguing that animals lacking the ability to efficiently and correctly remove dying cells should switch to a more global animal-wide system of stress resistance.Animals have many ways of protecting themselves against stress; for example, they can induce animal-wide, stress-protective pathways and they can kill damaged cells via apoptosis. We have discovered an unexpected regulatory relationship between these two types of stress responses. We find that  C. elegans, we find that these two responses are somehow linked: perturbing the mechanisms that allow cells to undergo apoptosis changes the whole animal's response to environmental stress. In fact, perturbing the apoptosis machinery in any way—through mutations that prevent apoptosis altogether, or through mutations that either slow or accelerate the clearance of dying cells—causes the animal to become more stress resistant. Together our findings raise the possibility that the animal may have a way of detecting defects in the normal programmed cell death pathway, and that in response it induces a new program that protects itself from a harsh environment.As an animal interacts with its environment, it invariably encounters stressful conditions such as extreme temperatures, drought, UV exposure and harmful xenobiotics. Since the ability to respond appropriately to stressful stimuli is paramount to survival, organisms have developed sophisticated stress response programs. Some stressful conditions cause damaged cells to commit suicide (undergo apoptosis), whereas others cause the entire organism to develop mechanisms to resist environmental stress. Studying the small roundworm  In nature, animals are constantly exposed to changing environmental conditions. In order to survive, organisms must weather normal and oftentimes extreme variations in temperature, water availability, salt levels, xenobiotics and other environmental factors. To cope, animals have developed mechanisms for stress protection. At the cellular level, DNA damage and other forms of stress can induce apoptosis to remove damaged cells; at the organismal level, stressful conditions can sometimes induce responses that make the entire animal more stress resistant C. elegans has evolved several stress-protective responses. These include aversive behaviors, such as avoidance of noxious stimuli, and the activation of alternative developmental programs, such as entry into the dauer state of diapause The nematode C. elegans, like other organisms, can protect itself against stress at the cellular level. For example, in C. elegans, germ cells undergo apoptosis in response to DNA damage from ionizing radiation In addition to coordinated stress responses at the organismal level, C. elegans caspase mutants, in which decisions about whether to complete the cell death program are made stochastically, a second mutation in an engulfment gene further reduces cell death As a fundamental process by which organisms remove unnecessary, abnormal or damaged cells, programmed cell death involves both cell killing via apoptosis and cell corpse removal via phagocytosis and degradation C. elegans  mutants, apoptotic cells are cleared approximately twice as fast as normal. We also showed that macrophages from progranulin null-mutant mice are able to engulf apoptotic cells more rapidly than are wild-type macrophages. Thus, progranulin, like mtm-1, abl-1 and srgp-1, is a negative regulator of programmed cell death clearance Previously, we showed that progranulin normally functions to regulate the rate of apoptotic cell engulfment during the process of programmed cell death pgrn-1(-) mutants exhibited an altered response to cellular stressors. We found that they did. However, unexpectedly, they demonstrated increased stress resistance. Even more surprisingly, we found that the same was true of mutations that perturb cell death in other ways, suggesting that a stress response pathway is activated when any part of the programmed cell death pathway does not proceed normally. Our findings reveal an unexpected link between mechanisms that control life-or-death decisions at the level of the individual cell and at the level of the entire animal.Given the close relationship between environmental stress and age-related disease, we asked whether pgrn-1(tm985) mutants were resistant to osmotic, heat and endoplasmic reticulum (ER) stress  , genotoxic stress (UV light) and pathogen exposure  mutants rescued or partially rescued the mutant stress resistance phenotypes  –C. In coenotypes –D. The phsp-4. HSP-4 is the nematode ortholog of mammalian grp78/BiP/HSP70, and is upregulated by heat and ER stress hsp-4::gfpP reporter levels  mutant animals also exhibited increased resistance to ER stress (pgrn-1(-); ced-3(n717) double mutants were no more stress-resistant than were either of the single mutants (pgrn-1 and ced-3 mutations may activate the same stress response pathway. ced-3(n717) mutants could also exhibit osmotic and heat stress resistance, albeit not as consistently as ER stress resistance  mutants did not exhibit resistance to paraquat or UV light  alleles, and these, in turn, were more resistant than the weak (n2438) allele  allele . These rC. elegans, ced-9 and ced-4 regulate the ability of ced-3 to activate programmed cell death ced-9 gain-of-function (gf) and ced-4 loss-of-function (lf or -) mutations are similar to ced-3(-) mutations in the sense that they impair programmed cell death, whereas ced-9(lf) mutations cause excessive cell death  due to uncontrolled activity of CED-4 and CED-3. We tested ced-9(n1950gf) and ced-4(n1162lf) single mutants, as well as a ced-4(n1162lf) ced-9(n2812lf) double mutant, for their responses to ER stress. We found that ced-9(gf) mutants were resistant to ER stress (ced-9(gf) mutations was not additive with that conferred by either pgrn-1(-) or ced-3(-) mutations, once again suggesting that these genes affect the same stress-response pathway (ced-4(lf) single mutants were resistant to ER stress at low doses of tunicamycin in one of two experiments (ced-9 for this stress resistance (ced-4 (and likely ced-3) may be genetically downstream of ced-9 in the stress-resistance pathway, as it is in the cell death pathway. However, since ced-4(lf) animals displayed an incomplete degree of stress resistance compared to ced-3(lf) and ced-9(gf) mutants, it remains possible that ced-4 is dispensable or redundant in this stress response pathway.In R stress . Further pathway . We alsoeriments , and thasistance . These fpgrn-1(-) or strong ced-3(-) mutant alleles. The engulfment mutants ced-1(e1735), ced-6(n1813), ced-7(n1892) and ced-2(e1752), ced-5(n1812), ced-10(n3246) exhibited resistance to ER stress at low doses of tunicamycin (1 µg/mL). However they exhibited variable responses to tunicamycin at higher doses (5 µg/mL), with ced-5, 7 and 10 mutants demonstrating ER stress resistance but not ced-1, 2 or 6 mutants (pgrn-1 and engulfment mutations were not more resistant than pgrn-1(-) alone, suggesting that these mutations could potentially induce a common ER stress-resistance pathway  and ced-2(e1752) mutants were resistant to osmotic stress  and one mutant allele of abi-1, ok171, were resistant to tunicamycin stress compared to wild type. Curiously, in some situations, abl-1 mutations actually reduced ER stress resistance. For example, abl-1(n1963) mutations alone have no visible effect on engulfment of apoptotic corpses; however, abl-1 mutations reduce the severity of the engulfment phenotype of ced-1(n2091) and ced-6(n2095) mutants abl-1(n1963) mutations reduced the level of ER stress resistance conferred by ced-1(n2091) and ced-6(n2095) mutations  nor unc-53(e404) mutants exhibited ER stress resistance phenotypes  mutants were resistant to ER stress at a low dose of tunicamycin but not at a high dose, similar to some of the engulfment mutants we tested such as ced-1, ced-2 and ced-6  and atf-6C. elegansxbp-1 mRNA by activated IRE-1 and the consequent upregulation of XBP-1 target genes, such as hsp-4, the nematode ortholog of mammalian grp78/BiP/HSP70 pgrn-1 mutants required ire-1 for ER stress resistance. We tested a pgrn-1; ire-1 double mutant and found that resistance in pgrn-1 mutants was dependent on ire-1  mutants were also generally unchanged compared to controls ; daf-2(e1370) double mutants was similar to that of daf-2(-) single mutants  pgrn-1(tm985) double mutants were no more stress resistant than were single daf-16(mu86) mutants  does not affect nuclear localization of DAF-16::GFP protein  into a pgrn-1(-) background AMpgrn-1(-); daf-16a strain was no more stress resistant than was the pgrn-1 mutant alone  mutant lifespan is no different than wild type ced-3(-) mutation significantly extended lifespan compared to wild type, ced-1 and ced-2 mutations did not ; pmk-1(km25) double mutants were no more resistant to ER stress than were pmk-1 single mutants. Thus, pmk-1 is required for the ER stress resistance induced by pgrn-1 mutations , for stress resistance and epistasis with pgrn-1(-). trf-1 mutants were not stress resistant compared to wild type and pgrn-1(-) did not require trf-1 for its stress resistance  and sortilin sistance . We alsosistance . Thus, apgrn-1(tm985), ced-3(n717) and ced-1(e1735) mutants to wild-type animals. This allowed us to assess 1) whether these strains have altered gene expression, and 2) whether their differentially expressed genes are shared, suggesting the involvement of a common pathway. In spite of different cell death phenotypes of these mutants, RNA-seq revealed that all three mutants down-regulated the same 95 genes and up-regulated the same 9 genes, a highly significant portion of the total transcriptome (p<10−16) , comparing day 1 adult p<10−16) . Of the p<10−16) . These fC. elegans programmed cell death in any of three ways—by inhibiting apoptosis, by impairing corpse clearance or by accelerating corpse clearance—all enhance resistance to certain environmental stressors. These mutations do not confer resistance to all cellular stressors, as pgrn-1 and ced-3 mutants exhibit normal sensitivity to UV light and oxidative stress.We have shown that mutations that impair hsp-4/BIP, whereas the cell-death-unrelated stressors Pseudomonas, UV and paraquat did not. However, hsp-4::gfpP was not induced by osmotic stress and pgrn-1(-) mutation did not increase basal hsp-4::gfpP expression levels. Thus, defects in cell death can do more to protect the animal than simply to induce the canonical UPR. They must generate a more multifaceted response that can maintain proteostasis.Several questions naturally follow from these findings. First, why are these mutants resistant to specific stressors? Tunicamycin is an N-linked glycosylation inhibitor that causes retention of translated proteins in the ER and induces the unfolded protein response. Similarly, heat and osmotic stress cause ER and/or cytosolic proteins to unfold and activate signaling programs that induce expression of heat shock proteins and other chaperones. Thus, it seems possible that cell death mutations all trigger a response specific for unfolded proteins, such as the ER unfolded protein response. Consistent with this, we found that the cell-death-related stressors tunicamycin and heat both activated the ER stress-response gene pgrn-1 mutants to resist the ER stressor tunicamycin. We found that this stress resistance requires an intact ire-1 gene, the MAP kinase PMK-1 and the transcription factor DAF-16/FOXO. We also identified a number of genes that are differentially regulated by all three of our mutants (compared to wild type), suggesting that they may achieve stress resistance by recruiting shared genes and/or pathways. It will be very interesting to explore these shared genes in future studies.The mechanism by which cell death mutations induce animal-wide stress resistance is not known. If different cell-death mutations affected different pathways, or the same pathway to different extents, then one would expect double mutants to be even more stress resistant than the individual single mutants. As this was not the case, it is possible that all of these mutations trigger the same stress response, though other interpretations remain possible. We identified some of the genes required for pgrn-1 mutants, daf-2 mutants are resistant to most environmental stressors. Thus, decreased DAF-2/insulin/IGF-1 signaling activates a genetic program that more generally elevates organismal resilience. daf-2 mutants appear to increase their resistance to ER stress by making the ire-1/xbp-1 pathway more efficient, possibly by activating stress-response transcription factors like DAF-16 that collaborate with ire-1/xbp-1 to induce new protective genes daf-2 mutants actually have reduced levels of expression of ire-1/xbp-1-regulated genes such as hsp-4/BIP. While this was not the case for pgrn-1 or ced-3 mutants, whose hsp-4::gfpP levels were not decreased, there are some unexpected similarities between the ER stress-resistance phenotypes of cell-death mutants and daf-2 mutants. First, the ER stress resistance phenotypes of pgrn-1 and daf-2 mutants require daf-16, either fully (pgrn-1 mutant) or partially (daf-2 mutant). Second, both ER stress responses are completely dependent on ire-1, yet in neither case is xbp-1 splicing increased. Additionally, the cell death mutants exhibited either no increase or only slight increases in hsp-4::gfpP expression, rather than the substantial increase one might expect if this branch of the ER pathway were constitutively active. Finally, the daf-2 and pgrn-1 mutant ER stress-resistance phenotypes are not additive. Thus the ER-stress resistance pathways activated by these mutations likely share at least some mechanistic features.In contrast to the selective stress resistance of It was striking that mutations that perturb programmed cell death in such different ways all had similar effects on environmental stress resistance. Why should mutations that lead to undead cells (apoptosis mutations), lingering corpses (engulfment mutations), and prematurely-engulfed corpses (progranulin mutations) all activate what appears to be (from genetic tests) the same stress resistance pathway? From an evolutionary perspective, this linkage may make sense. Presumably apoptosis evolved not only to sculpt tissues during development, but also to remove cells that are damaged and unable to perform their normal functions, or perhaps that are overtly harmful to the animal. Viewed in this way, impairments in programmed cell death could be interpreted by the organism as an inability to respond normally to stress. Perhaps, under these conditions, the animal uses an alternative, back-up system to survive; namely, the system we have described in this study. Specifically, animals could have developed a sensitive surveillance system that can detect abnormalities in cell death, and respond to them by activating another pathway that enhances their overall level of stress resistance. The existence of this type of alternative system could have increased animal fitness during evolution in turbulent or adverse environments.C. elegans, ced-10(-) mutations impair not only cell engulfment but also cell migration ced-1, has also been implicated in neuronal regulation of innate immunity While this model makes sense from an evolutionary perspective, other models are possible as well. For example, perhaps cell-death proteins, which act in a multi-step pathway to remove unwanted cells, also act together in a different pathway that has the effect of sensitizing the animal to various forms of stress. Non-cell death functions have, in fact, been described for cell-death effectors. In Arabidopsis may support the model that programmed cell death effectors can sensitize an animal to environmental stress. Arabidopsis can express a protein called RD21 that, like CED-3, is a cysteine protease, and, like progranulin, contains a granulin domain. Osmotic stress induces RD21 and, possibly as a consequence, leaf senescence. Interestingly, in response to stress RD21 undergoes a process of maturation in which its caspase domain cleaves and releases the granulin domain C. elegans, the two domains reside in different proteins but nevertheless act together to influence organismal stress resistance.A protein in the flowering plant ced-3, pgrn-1 and ced-1 have been implicated in organismal stress resistance, and these findings raise many interesting new questions about both mechanism and evolution.In summary, our findings indicate that programmed cell death effectors not only kill and remove individual cells, but also influence environmental stress resistance at the level of the whole animal. To our knowledge this is the first time that cell-death effectors like C. elegans were cultured at 20°C using standard procedures www.wormbase.org. The following strains were used:Unless otherwise indicated, AWK2ced-9(n1950gf) III; ced-3(n717) IVAWK74daf-16(mu86) I; ced-3(n717) IVAWK76daf-16::daf-16::gfp+Podr-1::RFP]daf-16(mu86) pgrn-1(tm985) I; muIs109[PAWK77daf-16::daf-16::gfp+Podr-1::RFP]daf-16(mu86) I; ced-3(n717) IV; muIs109[PAWK78ced-1(e1735) daf-16(mu86) IAWK80daf-16::daf-16::gfp+Podr-1::RFP]ced-1(e1735) daf-16(mu86) I; muIs109pgrn-1(tm985) I; muIs113[PCB404unc-53(e404) IICB936unc-73(e936) ICF1037daf-16(mu86) ICF1041daf-2(e1370) IIICF1934daf-16::daf-16::gfp+Podr-1::RFP]daf-16(mu86) I; muIs109[PCF2260hsp-4::gfp] VN2; zcIs4[PCF2473ire-1(ok799) IICF3050pgrn-1(tm985) ICF3165hsp-4-4::gfp] Vpgrn-1(tm985) I; zcIs4pgrn-1(tm985) I; muIs189 Line 1pgrn-1(tm986) I; muIs211[PCF3688egl-3::huPGRN::polycistronic mCherry+Podr-1::CFP] Line 2pgrn-1(tm986) I; muIs211[PCF3762ced-3(n1949) IVCF3802hsp-4::gfp] Vced-3(n717) IV; zcIs4[PCF3808trk-1(tm3985) XCF3809trk-1(tm4054) XCF3817pgrn-1(tm985) I; trk-1(tm3985) XCF3818pgrn-1(tm985) I; trk-1(tm4054) XCF3821trf-1(nr2014) IIICF3833pgrn-1(tm985) I; trf-1(nr2014) IIICF3879pgrn-1(tm985) I; pmk-1(km25) IVCF3881pgrn-1(tm985) I; ced-9(n1950gf) IIIFX494abi-1(tm494) IIIKU25pmk-1(km25) IVMT2547ced-4(n1162) IIIMT4433ced-6(n1813) IIIMT4982ced-7(n1892) IIIMT4770ced-9(n1950gf) IIIMT7384ced-4(n1162) ced-9(n2812lf) IIIMT16077ced-1(n2091) I; abl-1(n1963) XMT19956ced-6(n2095) III; abl-1(ok171) XOS4023pqn-41(ns294) IIIRB829abi-1(ok640) IIIXR1abl-1(ok171) XC. elegans pgrn-1 rescue construct, full-length pgrn-1a+TAA stop codon and its endogenous 0.5 kB upstream promoter were cloned into a Gateway polycistronic mCherry vector . The resulting plasmid (pgrn-1::pgrn-1+TAA::polycistronic mCherryP) expresses both progranulin and mCherry and functions as a full length rescuing construct when expressed in the pgrn-1 mutant. To generate a human progranulin rescue strain, the pan-neuronal egl-3 promoter (courtesy of K. Ashrafi lab) and the human progranulin cDNA sequence were cloned into a Gateway polycistronic mCherry vector (egl-1::human PGRN::polycistronic mCherryP).To generate a C. elegans. Stable monogenic lines were isolated and analyzed using Leica fluorescent, Zeiss Axioplan 2 or Nikon Spectral Confocal microscopes. Extrachromosomal arrays were integrated by UV irradiation by the method of C. Frank et al.The constructs were microinjected separately into the gonads of day 1 adult et al.2 UV light in a Stratalinker 1800 (Stratagene). Animals were then scored for survival every 24 hours. Pathogen stress was performed by transferring worms to plates seeded with P. aeruginosa or S. enterica starting at day 1 and scoring each subsequent day for survival. In all assays, animals that failed to move in response to a gentle touch with a metal pick were scored as dead.For thermal and osmotic stress assays on Day 1 worms, L4-stage animals were picked and grown at 20°C overnight. For thermal stress assays, worms were moved to a 35°C incubator for 12 hours and then scored for survival. Osmotic stress assays were performed by the method of Lamitina For ER stress assays, synchronized eggs were transferred to plates containing 0, 1, 2 or 5 µg/mL of tunicamycin . After 3 days, animals that developed to the L4 stage were quantified. Figures show fraction of animals that develop to L4 stage normalized to percent hatching on 0 µg/mL tunicamycin for each strain.Statistical analyses were performed in GraphPad Prism statistical package with tests as indicated in figure legends.ced-1(e1735), ced-2(e1752) and ced-3(n717) strains were grown at 20 degrees Celsius (C), then picked to fresh OP50 at the L4 stage and shifted to 25 degrees C. Subsequent lifespan analysis was done at this temperature. Animals were transferred every day to fresh plates until progeny production ceased. Animals that crawled off the plate, exploded, bagged, or became contaminated were censored. GraphPad Prism was used to calculate mean life spans and perform statistical analyses. P values were determined using log-rank (Mantel-Cox) statistics.Wild-type, C. elegans eggs were obtained by bleaching, then plated onto E. coli OP50 and allowed to develop at 20°C to day 1 of adulthood. At this point, positive controls were exposed to 5 mg/ml tunicamycin for 5 hours while all other worms were left untreated. After washing animals off plates, Trizol was added and samples were frozen in liquid nitrogen. Animals were lysed in a Mini-Beadbeater (Biospec products) for 10 minutes at the maximal setting. Total RNA was isolated using a phenol/chloroform extraction and DNA contamination was removed with DNA-free treatment (Ambion). cDNA was synthesized (iScript) using oligo (dT) primers and RT-PCR was performed using primers that amplify an ∼200 bp unspliced transcript and an ∼180 bp spliced transcript. Forward primer sequence: 5′ ctacgaagaagaagtcgtcgg 3′ and reverse primer sequence: 5′ ttcttgttgcgatccatgtg 3′. RT-PCR products were analyzed by running them out on a 3% agarose gel stained with ethidium bromide.hsp-4::gfpP transgene were anaesthetized on agarose pads containing 2.5 mM levamisole. Whole worm images were taken using a Retiga EXi Fast1394 CCD digital camera  using the 5× objective on a Zeiss Axioplan 2 compound microscope . Each image was taken so that the intestine was in focus and exposure time was calibrated to minimize number of saturated pixels for the set of animals. Images within each experiment were acquired using identical settings and exposure times to allow direct comparisons. Fluorescence intensity was measured by outlining the entire worm. Openlab 4.0.2 software  was then used to quantify total intensity of each pixel in the selected area. Measurements were obtained by subtracting the minimum intensity from the mean intensity and taking the average of these calculations for 8–10 animals per time-point.Animals expressing the pgrn-1(tm985), ced-3(n717), ced-1(e1735) and wild-type (N2E) using a phenol/chloroform extraction, and DNA contamination was removed with DNA-free treatment (Ambion). Samples were extracted in quadruplicates , for a total of 16 samples. Total RNA was quantified using the RiboGreen assay and RNA quality was checked using an Agilent Bioanalyzer (Agilent). RNA Integrity Numbers (RINs) were >8 in all the samples. Libraries for RNA-seq were prepared using the Illumina TruSeq library preparation protocol (Illumina Inc), multiplexed into a single pool and sequenced using an Illumina HiSeq 2500 sequencer across 4 lanes of 2 Rapid Run SR 1×50 flow cells. After demultiplexing, we obtained between 13 and 32 million reads per sample, each one 50 bases long. Quality control was performed on base qualities and nucleotide composition of sequences. Alignment to the C. elegans genome (ce10) was performed using the STAR spliced read aligner (PMID 23104886) with default parameters. Additional QC was performed after the alignment to examine the following: level of mismatch rate, mapping rate to the whole genome, repeats, chromosomes, and key transcriptomic regions . Between 92 and 93% of the reads mapped uniquely to the worm genome. Total counts of read-fragments aligned to candidate gene regions within the C. elegans reference gene annotation were derived using HTS-seq program and used as a basis for the quantification of gene expression. Only uniquely mapped reads were used for subsequent analyses. Following alignment and read quantification, we performed quality control using a variety of indices, including sample clustering, consistency of replicates, and average gene coverage. Differential expression analysis was performed using the EdgeR Bioconductor package (19910308), and differentially expressed genes were selected based on False Discovery Rate  estimated at ≤5%. Clustering and overlap analyses were performed using Bioconductor packages within the statistical environment R (www.r-project.org/). Gene Ontology annotation was performed using DAVID (david.abcc.ncifcrf.gov/). DAF-16 dependent genes were curated from published reports http://caprica.caltech.edu:9002/biomart/martview).Total RNA was isolated from each of the strains Figure S1C. elegans. Genes that normally promote cell death and engulfment are in green while those that normally inhibit them are shown in red. Genes tested in this study are in bold.Genetic pathways that regulate programmed cell death in apoptotic and engulfing cells in (TIFF)Click here for additional data file.Figure S2pgrn-1 does not confer oxidative, UV or pathogen stress resistance. Day 1 adult N2 or pgrn-1(tm985) animals were subjected to oxidative stress with 250 µM paraquat (A), genotoxic stress with 1200 J/m2 UV light (B) or pathogen stress by feeding P. aeruginosa (C). Worms were scored for survival at indicated times and analyses by log-rank Mantel-Cox test were performed. (A) p = 0.1, N>30 animals/strain. (B) p = 0.054, n>50 animals/strain. (C) p = 0.078, N>50 animals/strain. Results shown are representative of at least 2 experiments except in A, which was performed 3 times with p values of 0.0034, 0.01 and 0.125.Related to (TIFF)Click here for additional data file.Figure S3hsp-4::hsp-4::gfpP animals were incubated at 20°C (Control treated) or 35°C (Heat stress treated) for 4 hours then imaged on a Zeiss AxioImager. (B) hsp-4::hsp-4::gfpP animals were incubated at 20°C on agarose plates containing 0 µg/mL (Control treated) or 5 µg/mL (ER stress treated) tunicamycin. Twenty-four hours later, they were imaged on a Zeiss AxioImager. (C) hsp-4::hsp-4::gfpP animals were incubated at 20°C on agarose plates containing 51 mM NaCl (Control treated) or 400 mM NaCl (Osmotic stress treated). After 24 hours, they were imaged on a Zeiss AxioImager.Related to (TIFF)Click here for additional data file.Figure S4P. aeruginosa do not increase HSP-4::GFP levels. (A) hsp-4::hsp-4::gfpP animals were mock-treated (Control treated) or exposed to 1200 J/m2 of ultraviolet light (UV stress treated). After 5 hours at 20°C, the animals were imaged on a Zeiss AxioImager. (B) hsp-4::hsp-4::gfpP animals were placed onto agarose plates with either OP50 bacteria (Control treated) or P. aeruginosa PA14 (Pathogen treated) bacteria. After incubation for 24 hours at 20°C, animals were imaged on a Zeiss AxioImager. (C) hsp-4::hsp-4::gfpP animals were placed in M9 solution containing either vehicle alone (Control treated) or 200 mM paraquat (Oxidative stress treated) for 3 hours. The animals that were still alive at that time were imaged on a Zeiss AxioImager.Related to (TIFF)Click here for additional data file.Figure S5ced-3 does not confer oxidative or UV stress resistance. Day 1 adult N2 or ced-3(n717) animals were subjected to oxidative stress with 250 µM paraquat (A) or genotoxic stress with 1200 J/m2 UV light (B). Worms were scored for survival at indicated times and analyses by log-rank Mantel-Cox test were performed. (A) p>0.05, N = 20 animals/strain. (B) p>0.05, N>35 animals/strain.Related to (TIFF)Click here for additional data file.Figure S6ced-1(e1735) and ced-2(e1752) animals were exposed to 600 mM NaCl for 24 hours (A) or thermal stress at 35°C for 8 hours (B) and scored for survival. Error bars represent standard deviation. Statistical comparisons here are to N2 control (Student's t test or ANOVA with Bonferroni post-tests). *p<0.05, ***p<0.001. For additional statistical data, see Supplemental Related to (TIFF)Click here for additional data file.Figure S7ced-1(e2091) and ced-6(n2095) with or without abi-1(n2091 or ok171) in the background were tested for ER stress resistance by tunicamycin (TM) treatment. (B) Mutations in unc-53(e404) and unc-73(e936) were tested for ER stress resistance by tunicamycin treatment. (C) Mutations in pqn-41(ns924) with and without pgrn-1(tm985) in the background were tested for ER stress resistance by tunicamycin treatment. Results shown are representative of at least 2 experiments except in the case of (C) which was performed once. Error bars represent standard deviation. Statistical comparisons are to N2 control (ANOVA with Bonferroni post-tests). *p<0.05, **p<0.01, ***p<0.001. For additional statistical data, see Supplemental Related to (TIFF)Click here for additional data file.Figure S8xbp-1 mRNA is unchanged in pgrn-1(-), ced-1(-) or ced-3(-) mutants compared to wild type. Representative RT-PCR of xbp-1 from RNA isolated from day 1 animals. Arrows point to the unspliced and spliced xbp-1 bands and the loading control actin band. + TM refers to treatment with 5 mg/mL tunicamycin.Related to (TIFF)Click here for additional data file.Figure S9pgrn-1(-) or ced-3(-) mutants. Baseline levels of HSP-4::GFP in wild-type, pgrn-1(-) and ced-3(-) animals. Animals expressing hsp-4::hsp-4::gfpP in an otherwise wild-type, pgrn-1(tm985) or ced-3(n717) background were synchronized by washing adults and larvae away from eggs. L1 animals were collected from hatched embryos 1 hour later while L2, L3 and L4 larval stages were identified by size and anatomical landmarks. All animals were imaged and fluorescence quantified as in Experimental Methods.Related to (TIFF)Click here for additional data file.Figure S10pgrn-1(-), ced-1(-) or ced-3(-) mutants compared to wild type. Day 1 daf-16(-) adult animals expressing daf-16::daf-16::GFPP in an otherwise wild-type, daf-2(e1370), pgrn-1(tm985), ced-1(e1735) or ced-3(n717) background were imaged on a Zeiss AxioImager and DAF-16::GFP was scored as either nuclear or diffuse. Images shown are representative of 10 to 15 animals observed for each genotype except for ced-3(-) for which three animals were observed. Top row shows two representative animals and bottom row displays close-ups of these animals.Related to (TIFF)Click here for additional data file.Figure S11ced mutants. Survival of N2 Control, ced-3(n717), ced-1(e1735) and ced-2(e1752) animals was plotted across time. Compared to control, ced-3(-) animals lived significantly longer . N = 100 animals per strain.Lifespan analysis of (TIFF)Click here for additional data file.Figure S12C. elegans TRAF mutant, trf-1(nr2010), was tested for stress resistance. (B) Two alleles of the neurotrophin receptor trk-1 (tm3985 and tm4054) were tested for ER stress resistance. Error bars represent standard deviation. N = 50 animals in triplicate per strain per condition. *p<0.05, **p<0.01, ***p<0.001.TNFR/neurotrophin receptor-related genes are not resistant to ER stress. (A) A (TIFF)Click here for additional data file.Table S1pgrn-1 mutants are resistant to osmotic, thermal and unfolded protein stress. (A) Day-1 adult wild-type control animals and pgrn-1(-) mutants were exposed to osmotic stress with 600 mM NaCl for 24 hours and then scored for survival. Shown are mean survival ± SD and p value versus control (Student's t test). (B) Day-1 adult wild-type control animals and pgrn-1(-) mutants were exposed to thermal stress at 35°C for 8 hours and then scored for survival. Shown are mean survival ± SD and p value versus control (Student's t test). (C) Newly-laid wild-type control, pgrn-1(tm985) and pgrn-1(-); pgrn-1-rescue embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, those animals that had developed to L4 stage were counted. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (D) Newly laid embryos from wild-type control, pgrn-1(tm985) and pgrn-1(-); human PGRN-rescue (2 independent lines) were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests).Related to (DOCX)Click here for additional data file.Table S2ed-3(n717lf), ced-4(n162lf) and ced-9(n1950gf) mutations inhibit programmed cell death and confer stress resistance. (A) ER stress resistance is correlated with ced-3 allele strength. Newly laid embryos from wild-type control and ced-3 mutant alleles with graded abilities to inhibit programmed cell death  were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD is shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (B) Day-1 adult wild-type control animals and ced-3(-) mutants were exposed to osmotic stress with 600 mM NaCl for 24 hours or thermal stress at 35°C for 8 hours and scored for survival. Shown are mean survival ± SD and p value versus control (Student's t test). (C) A gain-of-function mutation in ced-9 increases stress resistance. Newly laid wild-type control, pgrn-1(tm985), ced-3(n717), ced-9(n1950gf), pgrn-1(-); ced-9(gf) and ced-3(n717); ced-9(gf) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (D) Newly laid wild-type control, pgrn-1(tm985), ced-4(n1162lf) and ced-4(n1162lf); ced-9(n2812lf) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests).Related to (DOCX)Click here for additional data file.Table S3pgrn-1(tm985), ced-1(e1735), ced-6(n1813) and ced-7(n1892) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. (B) Newly laid wild-type control, pgrn-1(tm985), ced-2(e1752), ced-5(n1812) and ced-10(n3246) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. (C) Day 1 adult wild-type control, ced-1(e1735), ced-6(n1813), ced-7(n1892), ced-2(e1752) and ced-5(n1812) mutants were exposed to osmotic stress with 600 mM NaCl for 24 hours or thermal stress at 35°C for 10 hours and then scored for survival. Shown are mean survival ± SD and p value versus control (ANOVA with Tukey post-test). (D) Newly laid wild-type control, pgrn-1(tm985), abl-1(ok171), abi-1(ok640) and abi-1(tm494) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (E) Newly laid wild-type control, pgrn-1(tm985), ced-1(n2091), ced-1(n2091); abi-1(n1963), ced-6(n2095), and ced-6(n2095); abi-1(n1963) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and ced-1 or ced-6 mutants are shown (ANOVA with Bonferroni post-tests). (F) Newly laid wild-type control, pgrn-1(tm985), unc-53(e404), and unc-73(e936) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (G) Newly laid wild-type control, pgrn-1(tm985), pqn-41(ns294), and pgrn-1(tm985); pqn-41(ns294) embryos were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD are shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests).Related to (DOCX)Click here for additional data file.Table S4pgrn-1(-) resistance to ER stress may be partially dependent on the UPR pathway, daf-16 and pmk-1. (A) Newly laid embryos from wild-type control, pgrn-1(tm985), ire-1(ok799) and pgrn-1(-); ire-1(-) mutants were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD is shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (B) Newly laid embryos from wild-type control, pgrn-1(tm985), daf-2(e1370), pgrn-1(-);daf-2(-), daf-16(mu86) and pgrn-1(-); daf-16(-) mutants were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD is shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (C) Newly laid embryos from wild-type control, pgrn-1(tm985), daf-16(mu86); muIs113 and pgrn-1(tm985); muIs113 mutants were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD is shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests). (D) Newly laid embryos from wild-type control, pgrn-1(tm985), pmk-1(km25) and pgrn-1(-); pmk-1(-) mutants were collected and placed onto plates with varying doses of tunicamycin. Three days later, the number of animals that had developed to L4 stage was determined. The fraction of animals that developed to L4 stage ± SD is shown. P value versus control and pgrn-1 mutant are shown (ANOVA with Bonferroni post-tests).Related to (DOCX)Click here for additional data file.Table S5pgrn-1(tm985), ced-3(n717), and ced-1(e1735) compared to N2E that are also regulated by daf-16. Hypergeometric distribution, p value<2×10−9.Supplementary dataset of differentially expressed genes. Each worksheet contains lists of genes differentially expressed with FDR<0.05. Lists include log2 fold-changes, p-values, and FDR for each gene. Fold-changes are color coded to denote up- or down-regulation . Please see first tab for contents of other tabs. Tab E shows genes differentially expressed in (XLSX)Click here for additional data file.