The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.     

Diverse Cell Type-Specific Mechanisms Localize G Protein-Coupled Receptors to Caenorhabditis elegans Sensory Cilia

The localization of signaling molecules such as G protein-coupled receptors (GPCRs) to primary cilia is essential for correct signal transduction. Detailed studies over the past decade have begun to elucidate the diverse sequences and trafficking mechanisms that sort and transport GPCRs to the ciliary compartment. However, a systematic analysis of the pathways required for ciliary targeting of multiple GPCRs in different cell types in vivo has not been reported. Here we describe the sequences and proteins required to localize GPCRs to the cilia of the AWB and ASK sensory neuron types in Caenorhabditis elegans. We find that GPCRs expressed in AWB or ASK utilize conserved and novel sequences for ciliary localization, and that the requirement for a ciliary targeting sequence in a given GPCR is different in different neuron types. Consistent with the presence of multiple ciliary targeting sequences, we identify diverse proteins required for ciliary localization of individual GPCRs in AWB and ASK. In particular, we show that the TUB-1 Tubby protein is required for ciliary localization of a subset of GPCRs, implying that defects in GPCR localization may be causal to the metabolic phenotypes of tub-1 mutants. Together, our results describe a remarkable complexity of mechanisms that act in a protein- and cell-specific manner to localize GPCRs to cilia, and suggest that this diversity allows for precise regulation of GPCR-mediated signaling as a function of external and internal context.     

Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport

The novel uncoupling proteins (UCP2-5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.     

Caenorhabditis elegans wsp-1 Regulation of Synaptic Function at the Neuromuscular Junction

The Rho GTPase members and their effector proteins, such as the Wiskott-Aldrich syndrome protein (WASP), play critical roles in regulating actin dynamics that affect cell motility, endocytosis, cell division, and transport. It is well established that Caenorhabditis elegans wsp-1 plays an essential role in embryonic development. We were interested in the role of the C. elegans protein WSP-1 in the adult nematode. In this report, we show that a deletion mutant of wsp-1 exhibits a strong sensitivity to the neuromuscular inhibitor aldicarb. Transgenic rescue experiments demonstrated that neuronal expression of WSP-1 rescued this phenotype and that it required a functional WSP-1 Cdc42/Rac interactive binding domain. WSP-1-GFP fusion protein was found localized presynaptically, immediately adjacent to the synaptic protein RAB-3. Strong genetic interactions with wsp-1 and other genes involved in different stages of synaptic transmission were observed as the wsp-1(gm324) mutation suppresses the aldicarb resistance seen in unc-13(e51), unc-11(e47), and snt-1 (md290) mutants. These results provide genetic and pharmacological evidence that WSP-1 plays an essential role to stabilize the actin cytoskeleton at the neuronal active zone of the neuromuscular junction to restrain synaptic vesicle release.     

Guidelines for monitoring autophagy in Caenorhabditis elegans

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.     

Defining Specificity Determinants of cGMP Mediated Gustatory Sensory Transduction in Caenorhabditis elegans

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.     

Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans

Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian G_o and G_q homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans. [BMB Reports 2014; 47(2): 80-85]     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)–treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L–like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease–causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms.     

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes

Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments.     

Mutation of the lbp-5 gene alters metabolic output in Caenorhabditis elegans

Intracellular lipid-binding proteins (LBPs) impact fatty acid homeostasis in various ways, including fatty acid transport into mitochondria. However, the physiological consequences caused by mutations in genes encoding LBPs remain largely uncharacterized. Here, we explore the metabolic consequences of lbp-5 gene deficiency in terms of energy homeostasis in Caenorhabditis elegans. In addition to increased fat storage, which has previously been reported, deletion of lbp-5 attenuated mitochondrial membrane potential and increased reactive oxygen species levels. Biochemical measurement coupled to proteomic analysis of the lbp-5(tm1618) mutant revealed highly increased rates of glycolysis in this mutant. These differential expression profile data support a novel metabolic adaptation of C. elegans, in which glycolysis is activated to compensate for the energy shortage due to the insufficient mitochondrial β-oxidation of fatty acids in lbp-5 mutant worms. This report marks the first demonstration of a unique metabolic adaptation that is a consequence of LBP-5 deficiency in C. elegans. [BMB Reports 2014; 47(1): 15-20]     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.     

Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion–deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.