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Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function. [ABSTRACT FROM AUTHOR]

Abstract: Adenylate kinase (AK; EC 2.7.4.3) is highly conserved across a wide range of organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens. While AK6 orthologs play important roles in the growth of yeast and worms, the physiological function of AK6 in A. thaliana is still unknown. In this study, we first cloned and expressed Arabidopsis adenylate kinase 6 (AAK6). Enzyme assays revealed that AAK6 has characteristic adenylate kinase properties, with ATP as the preferred phosphate donor and AMP as the best phosphate acceptor. A subcellular localization assay demonstrated that AAK6 had a predominant nuclear localization. Through biochemical purification and mass spectrometry analysis, a putative homolog of the S. cerevisiae Rps14 protein was identified as a partner of AAK6. Most importantly, we observed that aak6 T-DNA insertion mutants had decreased stem growth compared with wild-type plants. These data indicate that AAK6 exhibits adenylate kinase activity and is an essential growth factor in Arabidopsis. [Copyright &y& Elsevier]

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studying C. elegans sensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the βH-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending. [ABSTRACT FROM AUTHOR]

BACKGROUND: Auricularia polytricha (AP) is traditionally known for its medicinal properties, and linoleic acid (LA) is the prominent component in AP. OBJECTIVE: To understand the anti-aging and stress resistance mechanism induced by AP in Caenorhabditis elegans. METHODS: C. elegans (wild-type (N2), transgenic, and mutant strains) were treated with AP and LA and monitored for lifespan and stress resistance through physiological assays, fluorescence microscopy, and qPCR analysis. Molecular docking studies were employed to identify the interaction mode of LA with DAF-16 and SKN-1. RESULTS: Ethanol extract of AP (APE) was non-toxic and could induce an anti-aging mechanism, as it could extend the lifespan of nematode worms. This was dependent on PMK-1 and DAF-16 as APE could not extend the lifespan of these gene-specific mutants apart from extending the expression of these genes in wild-type nematodes, which was evident from qPCR analysis. LA, too, had a similar effect on the lifespan of wild-type and mutant worms, which further supported the findings. Molecular docking studies pointed to the role of DAF-16 and SKN-1 in regulating the effect of APE. APE also exhibited antioxidant and anti-inflammatory mechanisms as it significantly extended the lifespan in worms exposed to UV-B-induced oxidative stress, thereby showing a protective effect. APE could regulate SKN-1, which was evident from qPCR analysis and the fluorescence of skn-1:GFP transgenic strain. Further qPCR analysis of candidate regulatory genes exhibited antioxidant mechanisms induced by APE. CONCLUSION: APE was observed to induce anti-aging efficacy via SKN-1 and DAF-16. [ABSTRACT FROM AUTHOR]

Different biochars have diverse properties, with ambiguous effects on soil nematodes. This study investigated how aspen sawdust (ABC), bamboo powder (BBC), maize straw (MBC) and peanut-shell biochars (PBC) affected Caenorhabditis elegans via culture assays and RNA-seq analysis. The results showed that biochars derived from different agricultural materials varied significantly in physicochemical properties, and PBC produced more volatile organic compounds (VOCs) to attract C. elegans than ABC, BBC and MBC. Moreover, worms in ABC experienced the worst outcomes, while worms in PBC experienced milder impacts. Nematode body length decreased to 724.6 μm, 784.0 μm and 799.7 μm on average in ABC, BBC and MBC, respectively, compared to the control (1052 μm) and PBC treatments (960 μm). The brood size in ABC, MBC, BBC and PBC decreased 41.1%, 39.4%, 39.2% and 19.1% compared to the control, respectively. Furthermore, the molecular mechanisms of biochar-induced developmental effects on C. elegans were explored. Although several differentially expressed genes (DEGs) were different among the four biochars, worm phenotypic changes were mainly related to col genes (col-129; col-140; col-40; col-184), bli-6, sqt-3, perm-2/4, cdk-8, daf-16 and sod-1/2/5, which are associated with cuticle collagen synthesis, eggshell formation in postembryonic growth and rhythmic processes. Our study suggests that different properties of biochars could be crucial to soil nematodes, as well as the worms' biochemical changes are important for the health in agriculture soil. [ABSTRACT FROM AUTHOR]

Myo-Inositol (MI) has been shown to alleviate aging in Caenorhabditis (C). elegans. However, the mechanism by which MI alleviates aging remains unclear. In this study, we investigate whether MI can modulate the PI3K so as to attenuate the insulin/IGF-1 signaling (IIS) pathway and exert the longevity effect. The wild-type C. elegans and two mutants of AKT-1 and DAF-16 were used to explore the mechanism of MI so as to extend the lifespan, as well as to improve the health indexes of pharyngeal pumping and body bend, and an aging marker of autofluorescence in the C. elegans. We confirmed that MI could significantly extend the lifespan of C. elegans. MI also ameliorated the pharyngeal pumping and body bend and decreased autofluorescence. We further adopted the approach to reveal the loss-of-function mutants to find the signaling mechanism of MI. The functions of the lifespan-extending, health-improving, and autofluorescence-decreasing effects of MI disappeared in the AKT-1 and DAF-16 mutants. MI could also induce the nuclear localization of the DAF-16. Importantly, we found that MI could dramatically inhibit the phosphoinositide 3-kinase (PI3K) activity in a dose-dependent manner with an IC50 of 90.2 μM for the p110α isoform of the PI3K and 21.7 μM for the p110β. In addition, the downregulation of the PI3K expression and the inhibition of the AKT phosphorylation by MI was also obtained. All these results demonstrate that MI can inhibit the PI3K activity and downregulate the PI3K expression, and the attenuation of the IIS pathway plays a crucial role for MI in alleviating aging in C. elegans. [ABSTRACT FROM AUTHOR]

Pathogen recognition and the triggering of host innate immune system are critical to understanding pathogen-host interaction. Cellular surveillance systems have been identified as an important strategy for the identification of microbial infection. In the present study, using Bacillus thuringiensis-Caenorhabditis elegans as a model, we found an approach for surveillance systems to sense pathogens. We report that Bacillus thuringiensis Cry5Ba, a typical pore-forming toxin, caused mitochondrial damage and energy imbalance by triggering potassium ion leakage, instead of directly targeting mitochondria. Interestingly, we find C. elegans can monitor intracellular energy status to trigger innate immune responses via AMP-activated protein kinase (AMPK), secreting multiple effectors to defend against pathogenic attacks. Our study indicates that the imbalance of energy status is a prevalent side effect of pathogen infection. Furthermore, the AMPK-dependent surveillance system may serve as a practicable strategy for the host to recognize and defense against pathogens. Bacillus thuringiensis toxin Cry5Ba triggers potassium ion leakage, causing mitochondrial damage and energy imbalance. C. elegans can monitor this intracellular energy imbalance via AMP-activated protein kinase to trigger innate immune responses. [ABSTRACT FROM AUTHOR]

LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson's disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy.These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. [ABSTRACT FROM AUTHOR]

Cellular, small invertebrate and vertebrate models are a driving force in biogerontology studies. Using various models, such as yeasts, appropriate tissue culture cells, Drosophila, the nematode Caenorhabditis elegans and the mouse, has tremendously increased our knowledge around the relationship between diet, nutrient-response signaling pathways and lifespan regulation. In recent years, combinatorial drug treatments combined with mutagenesis, high-throughput screens, as well as multi-omics approaches, have provided unprecedented insights in cellular metabolism, development, differentiation, and aging. Scientists are, therefore, moving towards characterizing the fine architecture and cross-talks of growth and stress pathways towards identifying possible interventions that could lead to healthy aging and the amelioration of age-related diseases in humans. In this short review, we briefly examine recently uncovered knowledge around nutrient-response pathways, such as the Insulin Growth Factor (IGF) and the mechanistic Target of Rapamycin signaling pathways, as well as specific GWAS and some EWAS studies on lifespan and age-related disease that have enhanced our current understanding within the aging and biogerontology fields. We discuss what is learned from the rich and diverse generated data, as well as challenges and next frontiers in these scientific disciplines. [ABSTRACT FROM AUTHOR]

The secreted molecule unc-6/netrin is important for guiding axon projections and cell migrations, unc-5 and unc-40/DCC are identified as receptors for unc-6/netrin. The downstream factors of unc-6 receptors are beginning to be elucidated, and some key factors have been identified in various organisms. Here, we showed that SRC-1 interacts with the cytosolic domain of UNC-5 through its SH2 domain. This interaction also requires the intact kinase activity of SRC-1. Downregulation of src-1 by RNA interference decreases the biological processes initiated by the UNC-5 protein and decreases UNC-5 tyrosine phosphorylation. We also generated a chimeric protein consisting of the extracellular domain and transmembrane domain of UNC-5 and an intracellular domain of SRC-1. This fusion protein is able to partially rescue mutant phenotypes caused by unc-5 but not unc-6, unc-40, and unc-34. Our results support a model in which SRC-1 is required for UNC-5-induced axon repulsion and gonad migration signaling pathways and in which localizing SRC-1 activity to UNC-5 is crucial for proper signal transduction in response to unc-6/netrin. [ABSTRACT FROM AUTHOR]

Metformin, the most commonly prescribed anti‐diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti‐aging drug—the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose‐dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement. Enterohemorrhagic Escherichia coli (EHEC) induces formation of attaching and effacing lesions in the intestine. Here, Huang et al. use human intestinal cells and a C. elegans model of infection to show that the process is mediated by a host signaling pathway involving cyclin-dependent kinase CDK1 and formin CYK1. [ABSTRACT FROM AUTHOR]

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved "closure motif" region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation. Author summary: To segregate properly in meiosis, cohesion between replicated chromosomes must remain after the first meiotic cell division, so chromosomes can be held together until they finally separate in the second division. While the majority of organisms use centromeres to protect chromosome cohesion in the first division, the nematode worm C. elegans, which lacks single centromeres, instead protects cohesion only on a segment of the chromosome known as the "long arm". The long arm (and its complement, the short arm) are known to accumulate specific proteins and protein modifications, but it is not known how the short and long arms are first distinguished, nor how their separate functions are carried out. We report here that the chromosome axis protein HIM-3 and its modification by phosphorylation is important for ensuring the robust establishment of short and long arm functions. We show that phosphorylated HIM-3 partitions to the short arms after crossover recombination sites are designated, and HIM-3 mutants that mimic constitutive phosphorylation delay the normal establishment of the two complementary arm domains. Our findings reveal another layer of regulation to an outstanding mystery in chromosome biology. [ABSTRACT FROM AUTHOR]

Aging is an inevitable process characterized by the accumulation of degenerative damage, leading to serious diseases that affect human health. Studies on aging aim to develop pre‐protection or therapies to delay aging and age‐related diseases. A preventive approach is preferable to clinical treatment not only to reduce investment but also to alleviate pain in patients. Adjusting daily diet habits to improve the aging condition is a potentially attractive strategy. Fruits and vegetables containing active compounds that can effectively delay the aging process and reduce or inhibit age‐related degenerative diseases have been identified. The signaling pathways related to aging in Caenorhabditis elegans are evolutionarily conserved; thus, studying antiaging components by intervening senescence process may contribute to the prevention and treatment of age‐related diseases in humans. This review focuses on the effects of food‐derived extracts or purified substance on antiaging in nematodes, as well as the underlying mechanisms, on the basis of several major signaling pathways and key regulatory factors in aging. The aim is to provide references for a healthy diet guidance and the development of antiaging nutritional supplements. Finally, challenges in the use of C. elegans as the antiaging evaluation model are discussed, together with the development that potentially inspire novel strategies and research tools. [ABSTRACT FROM AUTHOR]

Oligomeric assembly of Amyloid-β (Aβ) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of Aβ oligomers could halt the disease progression. In this study, by using transgenic Caenorhabditis elegans model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber Cucumaria frondosa saponin with anti-cancer activity, on Aβ aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 µM significantly delayed the worm paralysis caused by Aβ aggregation as compared with control group. In addition, the number of Aβ plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of Aβ species in the transgenic C. elegans were significantly reduced upon treatment with frondoside A, whereas the level of Aβ monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of Aβ. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express Aβ. Taken together, these data suggested that low dose of frondoside A could protect against Aβ-induced toxicity by primarily suppressing the formation of Aβ oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-Aβ aggregation should be studied and elucidated in the future. [ABSTRACT FROM AUTHOR]

Slowing down aging-associated accumulation of molecular damage or its prevention represents a promising therapeutic paradigm to combat aging-related disease and death. While several chemical compounds extend lifespan in model organisms, their mechanism of action is often unknown, reducing their therapeutic potential. Using a systematic approach, here we characterize the impact of the GMP pathway on yeast lifespan and elucidate GMP synthesis inhibition as a lifespan extension mechanism. We further discover that proteasome activation extends lifespan in part through the GMP pathway. GMP synthesis inhibition exerts its lifespan extension effect independently of the canonical nutrient-sensing pathway regulating lifespan. Exposing longitudinally aging yeast cells to GMP pathway inhibition in an age-dependent manner, we demonstrate that the lifespan extension is facilitated by slowing, rather than reversing, the aging process in cells. Using a GUK1 mutant with lower GMP-to-GDP conversion activity, we observe lifespan extension, suggesting that reduced GDP level by itself can also extend yeast lifespan. These findings elucidate the involvement of nucleotide metabolism in the aging process. The existence of clinically-approved GMP pathway inhibitors elicits the potential of a new class of therapeutics for aging-related disorders. [ABSTRACT FROM AUTHOR]

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditis elegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo. Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context. [ABSTRACT FROM AUTHOR]

Aging research has experienced a burst of scientific efforts in the last decades as the growing ratio of elderly people has begun to pose an increased burden on the healthcare and pension systems of developed countries. Although many breakthroughs have been reported in understanding the cellular mechanisms of aging, the intrinsic and extrinsic factors that contribute to senescence on higher biological levels are still barely understood. The dog, Canis familiaris , has already served as a valuable model of human physiology and disease. The possible role the dog could play in aging research is still an open question, although utilization of dogs may hold great promises as they naturally develop age-related cognitive decline, with behavioral and histological characteristics very similar to those of humans. In this regard, family dogs may possess unmatched potentials as models for investigations on the complex interactions between environmental, behavioral, and genetic factors that determine the course of aging. In this review, we summarize the known genetic pathways in aging and their relevance in dogs, putting emphasis on the yet barely described nature of certain aging pathways in canines. Reasons for highlighting the dog as a future aging and gerontology model are also discussed, ranging from its unique evolutionary path shared with humans, its social skills, and the fact that family dogs live together with their owners, and are being exposed to the same environmental effects. [ABSTRACT FROM AUTHOR]

Understanding the plasticity, robustness and modularity of transcriptome expression to genetic and environmental conditions is crucial to deciphering how organisms adapt in nature. To test how genome architecture influences transcriptome profiles, we quantified expression responses for distinct temperature‐adapted genotypes of the nematode Caenorhabditis briggsae when exposed to chronic temperature stresses throughout development. We found that 56% of the 8,795 differentially expressed genes show genotype‐specific changes in expression in response to temperature (genotype‐by‐environment interactions, GxE). Most genotype‐specific responses occur under heat stress, indicating that cold vs. heat stress responses involve distinct genomic architectures. The 22 co‐expression modules that we identified differ in their enrichment of genes with genetic vs. environmental vs. interaction effects, as well as their genomic spatial distributions, functional attributes and rates of molecular evolution at the sequence level. Genes in modules enriched for simple effects of either genotype or temperature alone tend to evolve especially rapidly, consistent with disproportionate influence of adaptation or weaker constraint on these subsets of loci. Chromosome‐scale heterogeneity in nucleotide polymorphism, however, rather than the scale of individual genes predominates as the source of genetic differences among expression profiles, and natural selection regimes are largely decoupled between coding sequences and noncoding flanking sequences that contain cis‐regulatory elements. These results illustrate how the form of transcriptome modularity and genome structure contribute to predictable profiles of evolutionary change. [ABSTRACT FROM AUTHOR]

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defense genes, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection. [ABSTRACT FROM AUTHOR]

Membraneless organelles are distinct compartments within a cell that are not enclosed by a traditional lipid membrane and instead form through a process called liquid‐liquid phase separation. Examples of these non‐membrane‐bound organelles include nucleoli, stress granules, P bodies, pericentriolar material and germ granules. Many recent studies have used Caenorhabditis elegans germ granules, known as P granules, to expand our understanding of the formation of these unique cellular compartments. From this work, we know that proteins with intrinsically disordered regions (IDRs) play a critical role in the process of phase separation. IDR phase separation is further tuned through their interactions with RNA and through protein modifications such as phosphorylation and methylation. These findings from C elegans, combined with work done in other model organisms, continue to provide insight into the formation of membraneless organelles and the important role they play in compartmentalizing cellular processes. [ABSTRACT FROM AUTHOR]

The transforming growth factor-β (TGFβ) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGFβ ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGFβ signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGFβ receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGFβ receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGFβ receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGFβ pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer). [ABSTRACT FROM AUTHOR]

Nuclear hormone receptors (NHRs) are ligand-gated transcription factors that control adaptive host responses following recognition of specific endogenous or exogenous ligands. Although NHRs have expanded dramatically in C. elegans compared to other metazoans, the biological function of only a few of these genes has been characterized in detail. Here, we demonstrate that an NHR can activate an anti-pathogen transcriptional program. Using genetic epistasis experiments, transcriptome profiling analyses and chromatin immunoprecipitation-sequencing, we show that, in the presence of an immunostimulatory small molecule, NHR-86 binds to the promoters of immune effectors to activate their transcription. NHR-86 is not required for resistance to the bacterial pathogen Pseudomonas aeruginosa at baseline, but activation of NHR-86 by this compound drives a transcriptional program that provides protection against this pathogen. Interestingly, NHR-86 targets immune effectors whose basal regulation requires the canonical p38 MAPK PMK-1 immune pathway. However, NHR-86 functions independently of PMK-1 and modulates the transcription of these infection response genes directly. These findings characterize a new transcriptional regulator in C. elegans that can induce a protective host response towards a bacterial pathogen. [ABSTRACT FROM AUTHOR]

The small GTPase RhoA plays a crucial role in the regulation of neuronal signalling to generate behaviour. In the developing nervous system RhoA is known to regulate the actin cytoskeleton, however the effectors of RhoA-signalling in adult neurons remain largely unidentified. We have previously shown that activation of the RhoA ortholog (RHO-1) in C. elegans cholinergic motor neurons triggers hyperactivity of these neurons and loopy locomotion with exaggerated body bends. This is achieved in part through increased diacylglycerol (DAG) levels and the recruitment of the synaptic vesicle protein UNC-13 to synaptic release sites, however other pathways remain to be identified. Dopamine, which is negatively regulated by the dopamine re-uptake transporter (DAT), has a central role in modulating locomotion in both humans and C. elegans. In this study we identify a new pathway in which RHO-1 regulates locomotory behaviour by repressing dopamine signalling, via DAT-1, linking these two pathways together. We observed an upregulation of dat-1 expression when RHO-1 is activated and show that loss of DAT-1 inhibits the loopy locomotion phenotype caused by RHO-1 activation. Reducing dopamine signalling in dat-1 mutants through mutations in genes involved in dopamine synthesis or in the dopamine receptor DOP-1 restores the ability of RHO-1 to trigger loopy locomotion in dat-1 mutants. Taken together, we show that negative regulation of dopamine signalling via DAT-1 is necessary for the neuronal RHO-1 pathway to regulate locomotion. [ABSTRACT FROM AUTHOR]

Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL–MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL–MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL–MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL–MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL–MLT and CDC-42–SID-3 activities in the epidermis. [ABSTRACT FROM AUTHOR]

The Pam/Highwire/RPM-1 (PHR) proteins are conserved intracellular signaling hubs that regulate synapse formation and axon termination. The C. elegans PHR protein, called RPM-1, acts as a ubiquitin ligase to inhibit the DLK-1 and MLK-1 MAP kinase pathways. We have identified several kinases that are likely to form a new MAP kinase pathway that suppresses synapse formation defects, but not axon termination defects, in the mechanosensory neurons of rpm-1 mutants. This pathway includes: MIG-15 (MAP4K), NSY-1 (MAP3K), JKK-1 (MAP2K) and JNK-1 (MAPK). Transgenic overexpression of kinases in the MIG-15/JNK-1 pathway is sufficient to impair synapse formation in wild-type animals. The MIG-15/JNK-1 pathway functions cell autonomously in the mechanosensory neurons, and these kinases localize to presynaptic terminals providing further evidence of a role in synapse development. Loss of MIG-15/JNK-1 signaling also suppresses defects in habituation to repeated mechanical stimuli in rpm-1 mutants, a behavioral deficit that is likely to arise from impaired glutamatergic synapse formation. Interestingly, habituation results are consistent with the MIG-15/JNK-1 pathway functioning as a parallel opposing pathway to RPM-1. These findings indicate the MIG-15/JNK-1 pathway can restrict both glutamatergic synapse formation and short-term learning. [ABSTRACT FROM AUTHOR]

The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode Caenorhabditis elegans, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in C. elegans grown in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in C. elegans, and suggests a mechanism for cross-talk between stress responses. [ABSTRACT FROM AUTHOR]

Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 genes alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Several types of associative learning are dependent upon the presence or absence of food, and are crucial for the survival of most animals. Target of rapamycin (TOR), a kinase which exists as a component of two complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2), is known to act as a nutrient sensor in numerous organisms. However, the in vivo roles of TOR signaling in the nervous system remain largely unclear, partly because its multifunctionality and requirement for survival make it difficult to investigate. Here, using pharmacological inhibitors and genetic analyses, we show that TORC1 and TORC2 contribute to associative learning between salt and food availability in the nematode Caenorhabditis elegans in a process called taste associative learning. Worms migrate to salt concentrations experienced previously during feeding, but they avoid salt concentrations experienced under starvation conditions. Administration of the TOR inhibitor rapamycin causes a behavioral defect after starvation conditioning. Worms lacking either RICT-1 or SINH-1, two TORC2 components, show defects in migration to high salt levels after learning under both fed and starved conditions. We also analyzed the behavioral phenotypes of mutants of the putative TORC1 substrate RSKS-1 (the C. elegans homolog of the mammalian S6 kinase S6K) and the putative TORC2 substrates SGK-1 and PKC-2 (homologs of the serum and glucocorticoid-induced kinase 1, SGK1, and protein kinase C-α, PKC-α, respectively) and found that neuronal RSKS-1 and PKC-2, as well as intestinal SGK-1, are involved in taste associative learning. Our findings shed light on the functions of TOR signaling in behavioral plasticity and provide insight into the mechanisms by which information sensed in the intestine affects the nervous system to modulate food-searching behaviors. [ABSTRACT FROM AUTHOR]

Spatial arrangement of neurite branching is instructed by both attractive and repulsive cues. Here we show that in C. elegans, the Wnt family of secreted glycoproteins specify neurite branching sites in the PLM mechanosensory neurons. Wnts function through MIG-1/Frizzled and the planar cell polarity protein (PCP) VANG-1/Strabismus/Vangl2 to restrict the formation of F-actin patches, which mark branching sites in nascent neurites. We find that VANG-1 promotes Wnt signaling by facilitating Frizzled endocytosis and genetically acts in a common pathway with arr-1/β-arrestin, whose mutation results in defective PLM branching and F-actin patterns similar to those in the Wnt, mig-1 or vang-1 mutants. On the other hand, the UNC-6/Netrin pathway intersects orthogonally with Wnt-PCP signaling to guide PLM branch growth along the dorsal-ventral axis. Our study provides insights for how attractive and repulsive signals coordinate to sculpt neurite branching patterns, which are critical for circuit connectivity. [ABSTRACT FROM AUTHOR]

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. [ABSTRACT FROM AUTHOR]

Protein phosphorylation underlies cellular response pathways across eukaryotes and is governed by the opposing actions of phosphorylating kinases and de-phosphorylating phosphatases. While kinases and phosphatases have been extensively studied, their organization and the mechanisms by which they balance each other are not well understood. To address these questions we performed quantitative analyses of large-scale 'omics' datasets from yeast, fly, plant, mouse and human. We uncovered an asymmetric balance of a previously-hidden scale: Each organism contained many different kinase genes, and these were balanced by a small set of highly abundant phosphatase proteins. Kinases were much more responsive to perturbations at the gene and protein levels. In addition, kinases had diverse scales of phenotypic impact when manipulated. Phosphatases, in contrast, were stable, highly robust and flatly organized, with rather uniform impact downstream. We validated aspects of this organization experimentally in nematode, and supported additional aspects by theoretic analysis of the dynamics of protein phosphorylation. Our analyses explain the empirical bias in the protein phosphorylation field toward characterization and therapeutic targeting of kinases at the expense of phosphatases. We show quantitatively and broadly that this is not only a historical bias, but stems from wide-ranging differences in their organization and impact. The asymmetric balance between these opposing regulators of protein phosphorylation is also common to opposing regulators of two other post-translational modification systems, suggesting its fundamental value. [ABSTRACT FROM AUTHOR]

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle. [ABSTRACT FROM AUTHOR]

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2–JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration. [ABSTRACT FROM AUTHOR]

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, in this paper we use tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis. CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function we also show that the PAR complex functions in tip formation and orientation. Additionally, we find that the sole C. elegans Eph receptor, VAB-1, functions during this process in an Ephrin-independent manner. Using epistasis analysis, we find that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation. [ABSTRACT FROM AUTHOR]

To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation. [ABSTRACT FROM AUTHOR]

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα–LGN–NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation. [ABSTRACT FROM AUTHOR]

Regulation of synaptic AMPA receptor levels is a major mechanism underlying homeostatic synaptic scaling. While in vitro studies have implicated several molecules in synaptic scaling, the in vivo mechanisms linking chronic changes in synaptic activity to alterations in AMPA receptor expression are not well understood. Here we use a genetic approach in C. elegans to dissect a negative feedback pathway coupling levels of the AMPA receptor GLR-1 with its own transcription. GLR-1 trafficking mutants with decreased synaptic receptors in the ventral nerve cord (VNC) exhibit compensatory increases in glr-1 mRNA, which can be attributed to increased glr-1 transcription. Glutamatergic transmission mutants lacking presynaptic eat-4/VGLUT or postsynaptic glr-1, exhibit compensatory increases in glr-1 transcription, suggesting that loss of GLR-1 activity is sufficient to trigger the feedback pathway. Direct and specific inhibition of GLR-1-expressing neurons using a chemical genetic silencing approach also results in increased glr-1 transcription. Conversely, expression of a constitutively active version of GLR-1 results in decreased glr-1 transcription, suggesting that bidirectional changes in GLR-1 signaling results in reciprocal alterations in glr-1 transcription. We identify the CMK-1/CaMK signaling axis as a mediator of the glr-1 transcriptional feedback mechanism. Loss-of-function mutations in the upstream kinase ckk-1/CaMKK, the CaM kinase cmk-1/CaMK, or a downstream transcription factor crh-1/CREB, result in increased glr-1 transcription, suggesting that the CMK-1 signaling pathway functions to repress glr-1 transcription. Genetic double mutant analyses suggest that CMK-1 signaling is required for the glr-1 transcriptional feedback pathway. Furthermore, alterations in GLR-1 signaling that trigger the feedback mechanism also regulate the nucleocytoplasmic distribution of CMK-1, and activated, nuclear-localized CMK-1 blocks the feedback pathway. We propose a model in which synaptic activity regulates the nuclear localization of CMK-1 to mediate a negative feedback mechanism coupling GLR-1 activity with its own transcription. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension. [ABSTRACT FROM AUTHOR]

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. [ABSTRACT FROM AUTHOR]

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii. [ABSTRACT FROM AUTHOR]

Zuccagnia punctata Cav. (Fabaceae), commonly called jarilla macho or pus-pus, is being used in traditional medicine as an antiseptic, anti-inflammatory and to relieve muscle and bone pain. The aim of this work was to study the anthelmintic effects of three structurally related flavonoids present in aerial parts of Z. punctata Cav. The biological activity of the flavonoids 7-hydroxyflavanone (HF), 3,7-dihydroxyflavone (DHF) and 2´,4´-dihydroxychalcone (DHC) was examined in the free-living nematode Caenorhabditis elegans. Our results showed that among the assayed flavonoids, only DHC showed an anthelmintic effect and alteration of egg hatching and larval development processes in C. elegans. DHC was able to kill 50% of adult nematodes at a concentration of 17 μg/mL. The effect on larval development was observed after 48 h in the presence of 25 and 50 μg/mL DHC, where 33.4 and 73.4% of nematodes remained in the L3 stage or younger. New therapeutic drugs with good efficacy against drug-resistant nematodes are urgently needed. Therefore, DHC, a natural compound present in Z. punctata, is proposed as a potential anthelmintic drug. [ABSTRACT FROM AUTHOR]

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. [ABSTRACT FROM AUTHOR]

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation. [ABSTRACT FROM AUTHOR]

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis. [ABSTRACT FROM AUTHOR]

Background: Proving the efficacy and corresponding mode of action of herbal supplements is a difficult challenge for evidence-based herbal therapy. A major hurdle is the complexity of herbal preparations, many of which combine multiple herbs, particularly when the combination is assumed to be vitally important to the effectiveness of the herbal therapy. This issue may be addressed through the use of contemporary methodology and validated animal models. Methods and Principal Findings: In this study, two commonly used traditional herbal formulas, Shi Quan Da Bu Tang (SQDB) and Huo Luo Xiao Ling Dan (HLXL) were evaluated using a survival assay and oxidative stress biomarkers in a well-established C. elegans model of aging. HLXL is an eleven herb formula modified from a top-selling traditional herbal formula for the treatment of arthritic joint pain. SQDB consists of ten herbs often used for fatigue and energy, particularly in the aged. We demonstrate here that SQDB significantly extend life span in a C. elegans model of aging. Among all individual herbs tested, two herbs Cinnamomum cassia bark (Chinese pharmaceutical name: Cinnamomi Cortex, CIN) and Panax ginseng root (Chinese pharmaceutical name: Ginseng Radix, GS) significantly extended life span in C. elegans. CIN in both SQDB and HLXL formula extended life span via modulation of multiple longevity assurance genes, including genes involved in insulin signaling and stress response pathways. All the life-span-extending herbs (SQDB, CIN and GS) also attenuated levels of H2O2 and enhanced small heat shock protein expression. Furthermore, the life span-extending herbs significantly delayed human amyloid beta (Aβ)-induced toxicity in transgenic C. elegans expressing human Ab. Conclusion/Significance: These results validate an invertebrate model for rapid, systematic evaluation of commonly used Chinese herbal formulations and may provide insight for designing future evidence-based herbal therapy(s). [ABSTRACT FROM AUTHOR]

Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF-16, and the energy sensor, AMP-activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health-related benefits of dietary restriction. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis. [ABSTRACT FROM AUTHOR]