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Phoresy is an interspecies interaction that facilitates spatial dispersal by attaching to a more mobile species. Hitchhiking species have evolved specific traits for physical contact and successful phoresy, but the regulatory mechanisms involved in such traits and their evolution are largely unexplored. The nematode Caenorhabditis elegans displays a hitchhiking behavior known as nictation during its stress-induced developmental stage. Dauer-specific nictation behavior has an important role in natural C. elegans populations, which experience boom-and-bust population dynamics. In this study, we investigated the nictation behavior of 137 wild C. elegans strains sampled throughout the world. We identified species-wide natural variation in nictation and performed a genome-wide association mapping. We show that the variants in the promoter of nta-1 , encoding a putative steroidogenic enzyme, underlie differences in nictation. This difference is due to the changes in nta-1 expression in glial cells, which implies that glial steroid metabolism regulates phoretic behavior. Population genetic analysis and geographic distribution patterns suggest that balancing selection maintained two nta-1 haplotypes that existed in ancestral C. elegans populations. Our findings contribute to further understanding of the molecular mechanism of species interaction and the maintenance of genetic diversity within natural populations., Competing Interests: Competing interests statement:The authors declare no competing interest.

The transforming growth factor beta (TGF-β) signaling pathway is implicated in various physiological processes, including neuronal functions. In the nematode Caenorhabditis elegans , TGF-β plays an important role in behaviors like nictation, a behavior crucial for dispersal. Through forward mutagenesis screening, we identified tax -4, a cyclic nucleotide-gated channel, as a suppressor of nictation defects in daf -7 mutants. Further investigation revealed the role of tax -4 in gustatory neurons. Furthermore, our findings indicate that the TAX-2/ TAX-4 heteromeric channel is likely to be involved in the regulation of nictation. These observations provide insights into the complexity of neuronal regulation and offer potential implications for understanding TGF-β dysregulation, which is common in many human brain diseases.

Under adverse environmental conditions, nematodes arrest into dauer, an alternative developmental stage for diapause. Dauer endures unfavorable environments and interacts with host animals to access favorable environments, thus playing a critical role in survival. Here, we report that in Caenorhabditis elegans, daf-42 is essential for development into the dauer stage, as the null mutant of daf-42 exhibited a "no viable dauer" phenotype in which no viable dauers were obtained in any dauer-inducing conditions. Long-term time lapse microscopy of synchronized larvae revealed that daf-42 is involved in developmental changes from the pre-dauer L2d stage to the dauer stage. daf-42 encodes large, disordered proteins of various sizes that are expressed in and secreted from the seam cells within a narrow time window shortly before the molt into dauer stage. Transcriptome analysis showed that the transcription of genes involved in larval physiology and dauer metabolism is highly affected by the daf-42 mutation. Contrary to the notion that essential genes that control the life and death of an organism may be well conserved across diverse species, daf-42 is an evolutionarily young gene conserved only in the Caenorhabditis genus. Our study shows that dauer formation is a vital process that is controlled not only by conserved genes but also by newly emerged genes, providing important insights into evolutionary mechanisms., Competing Interests: Conflicts of interest statement The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Animals exhibit phenotypic plasticity through the interaction of genes with the environment, and little is known about the genetic factors that change synaptic function at different developmental stages. Here, we investigated the genetic determinants of how animal's sensitivity to drugs that alter synaptic activity is regulated at a specific developmental stage using the free-living nematode Caenorhabditis elegans. C. elegans enters the stress-resistant dauer larval stage under harsh conditions. Although dauer is known to have reduced permeability and increased resistance to most known exogenous chemicals, we discovered that dauer is hypersensitive to a cholinesterase inhibitor, aldicarb. To investigate genes regulating dauer-specific acetylcholine transduction, we first screened for aldicarb-resistant mutations in dauer and then performed a secondary screen to rule out aldicarb-resistant mutations that also affect adults. We isolated 2 different mutations of a single gene called cyp-34A4 or dach-1 encoding a cytochrome P450. In the nondauer stages, dach-1 is mainly expressed in the intestine, but its expression is robustly increased in the epidermis of dauers. By tissue-specific rescue experiments, we found that dach-1 modulates aldicarb sensitivity in a cell nonautonomous manner. In addition, dach-1 plays pleiotropic functions in dauers by regulating quiescence and surviving heat shock and hyperosmolar stress. Our study reveals novel functions of the cytochrome P450 in synaptic and physiological changes during the developmental plasticity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Cilia are important for the interaction with environments and the proper function of tissues. While the basic structure of cilia is well conserved, ciliated cells have various functions. To understand the distinctive identities of ciliated cells, the identification of cell-specific proteins and its regulation is essential. Here, we report the mechanism that confers a specific identity on IL2 neurons in Caenorhabditis elegans, neurons important for the dauer larva-specific nictation behavior. We show that DAF-19M, an isoform of the sole C. elegans RFX transcription factor DAF-19, heads a regulatory subroutine, regulating target genes through an X-box motif variant under the control of terminal selector proteins UNC-86 and CFI-1 in IL2 neurons. Considering the conservation of DAF-19M module in IL2 neurons for nictation and in male-specific neurons for mating behavior, we propose the existence of an evolutionarily adaptable, hard-wired genetic module for distinct behaviors that share the feature "recognizing the environment.", Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although multiple determinants for establishing polarity in membranes of epithelial cells have been identified, the mechanism for maintaining apicobasal polarity is not fully understood. Here, we show that the conserved Hippo kinase pathway plays a role in the maintenance of apicobasal polarity in the developing intestine of Caenorhabditis elegans We screened suppressors of the mutation in wts-1 -the gene that encodes the LATS kinase homolog, deficiency of which leads to disturbance of the apicobasal polarity of the intestinal cells and to eventual death of the organism. We identified several alleles of yap-1 and egl-44 that suppress the effects of this mutation. yap-1 encodes a homolog of YAP/Yki, and egl-44 encodes a homolog of TEAD/Sd. WTS-1 bound directly to YAP-1 and inhibited its nuclear accumulation in intestinal cells. We also found that NFM-1, which is a homolog of NF2/Merlin, functioned in the same genetic pathway as WTS-1 to regulate YAP-1 to maintain cellular polarity. Transcriptome analysis identified several target candidates of the YAP-1-EGL-44 complex including TAT-2, which encodes a putative P-type ATPase. In summary, we have delineated the conserved Hippo pathway in C. elegans consisting of NFM-1-WTS-1-YAP-1-EGL-44 and proved that the proper regulation of YAP-1 by upstream NFM-1 and WTS-1 is essential for maintenance of apicobasal membrane identities of the growing intestine., (Copyright © 2019 by the Genetics Society of America.)

Guidance molecules, receptors, and downstream signaling pathways involved in the asymmetric neuronal cell migration and process outgrowth have been identified from genetic studies using model organisms, most of which are evolutionarily conserved. In the nematode Caenorhabditis elegans , the roles of Wnt ligands and their receptors in the polarization of specific sets of neurons along the anterior-posterior (A-P) body axis have been well elucidated, but their downstream effectors are relatively unknown. Here, we report yap-1 , encoding an evolutionarily conserved transcriptional co-activator, as a novel player in the Wnt-mediated asymmetric development of specific neurons in C. elegans We found that the loss of yap-1 activity failed to restrict the dendritic extension of ALM neurons to the anterior orientation, which is similar to the phenotype caused by defective cwn-1 and cwn-2 Wnt gene activities. Cell-specific rescue experiments showed that yap-1 acts in the cell autonomous manner to polarize ALM dendrites. We also found that subcellular localization of YAP-1 was spatio-temporally regulated. The loss of yap-1 in Wnt-deficient mutants did not increase the severity of the ALM polarity defect of the mutants. Wnt-deficient animals displayed abnormal subcellular localization of YAP-1 in touch receptor neurons, suggesting that yap-1 may act downstream of the cwn-1/cwn-2 Wnt ligands for the ALM polarization process. Together, we have identified a new role for YAP-1 in neuronal development and our works will contribute to further understanding of intracellular events in neuronal polarization during animal development., (Copyright © 2018 Lee et al.)

Phoresy is a widespread form of commensalism that facilitates dispersal of one species through an association with a more mobile second species. Dauer larvae of the nematode Caenorhabditis elegans exhibit a phoretic behavior called nictation, which could enable interactions with animals such as isopods or snails. Here, we show that natural C. elegans isolates differ in nictation. We use quantitative behavioral assays and linkage mapping to identify a genetic locus (nict-1) that mediates the phoretic interaction with terrestrial isopods. The nict-1 locus contains a Piwi-interacting small RNA (piRNA) cluster; we observe that the Piwi Argonaute PRG-1 is involved in the regulation of nictation. Additionally, this locus underlies a trade-off between offspring production and dispersal. Variation in the nict-1 locus contributes directly to differences in association between nematodes and terrestrial isopods in a laboratory assay. In summary, the piRNA-rich nict-1 locus could define a novel mechanism underlying phoretic interactions.Nematodes use a characteristic set of movements, called nictation, to hitchhike on more mobile animals. Here, Lee et al. identify a genetic locus in the nematode Caenorhabditis elegans that underlies nictation and contributes to successful hitchhiking, but at expense of reduced offspring production.

Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle ( i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cells surviving crisis are often tumorigenic and their telomeres are commonly maintained through the reactivation of telomerase. However, surviving cells occasionally activate a recombination-based mechanism called alternative lengthening of telomeres (ALT). Here we establish stably maintained survivors in telomerase-deleted Caenorhabditis elegans that escape from sterility by activating ALT. ALT survivors trans-duplicate an internal genomic region, which is already cis-duplicated to chromosome ends, across the telomeres of all chromosomes. These 'Template for ALT' (TALT) regions consist of a block of genomic DNA flanked by telomere-like sequences, and are different between two genetic background. We establish a model that an ancestral duplication of a donor TALT region to a proximal telomere region forms a genomic reservoir ready to be incorporated into telomeres on ALT activation.

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Although many critical roles of the RUNX family proteins have already been identified, little attention has been given to how these proteins interact with other factors. Elucidating RUNX protein interactions will help extend our understanding of their roles in normal development and tumorigenesis. In this study, we performed large-scale RNAi screening to identify genes that genetically interact with rnt-1, the sole homolog of RUNX protein in the nematode Caenorhabditis elegans. To this end, we took advantage of the fact that C. elegans can survive a severe loss of RNT-1 function with only mild phenotypes, and we looked for genes that caused a synthetic phenotype in the rnt-1 mutant background. We identified seven genes, three of which (cdk-8, cic-1, and sur-2) are involved in transcription, two of which (pgp-2 and cct-5) are involved in stress response, and two of which (D2045.7 and W09D10.4) are involved in signaling cascades, according to their functional gene ontology terms. We further confirmed that the CDK8-containing mediator complex genetically interacts with RNT-1 by showing that knockdown of each component of the CDK8 mediator complex caused a synthetic phenotype, that is, the exploded intestine through the vulva (Eiv) phenotype, in the rnt-1 mutant background. We also identified a putative target gene, acs-4, which is regulated by the RNT-1 and CDK8 mediator complex. Our results strengthen the notion that the CDK8 mediator complex may also act together with RUNX proteins in mammals.

Background: Dendrites often display remarkably complex and diverse morphologies that are influenced by developmental and environmental cues. Neuroplasticity in response to adverse environmental conditions entails both hypertrophy and resorption of dendrites. How dendrites rapidly alter morphology in response to unfavorable environmental conditions is unclear. The nematode Caenorhabditis elegans enters into a stress-resistant dauer larval stage in response to an adverse environment., Results: Here we show that the IL2 bipolar sensory neurons undergo dendrite arborization and axon remodeling during dauer development. When dauer larvae are returned to favorable environmental conditions, animals resume reproductive development and IL2 dendritic branches retract, leaving behind remnant branches in postdauer L4 and adult animals. The C. elegans furin homolog KPC-1 is required for dauer IL2 dendritic arborization and dauer-specific nictation behavior. KPC-1 is also necessary for dendritic arborization of PVD and FLP sensory neurons. In mammals, furin is essential, ubiquitously expressed, and associated with numerous pathologies, including neurodegenerative diseases. While broadly expressed in C. elegans neurons and epithelia, KPC-1 acts cell autonomously in IL2 neurons to regulate dauer-specific dendritic arborization and nictation., Conclusions: Neuroplasticity of the C. elegans IL2 sensory neurons provides a paradigm to study stress-induced and reversible dendritic branching, and the role of environmental and developmental cues in this process. The newly discovered role of KPC-1 in dendrite morphogenesis provides insight into the function of proprotein convertases in nervous system development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Caenorhabditis elegans microRNAs (miRNAs) bind to partially complementary sequences in the 3' untranslated region of target mRNAs, resulting in translational repression through mRNA destabilization. High-throughput sequencing of RNA cleavage fragments was performed to directly detect miRNA-directed cleavage targets in adult stage C. elegans. From this analysis, we found that miR-249 directed the cleavage of the ZK637.6 transcript with extensive and evolutionarily conserved complementarity in nematode. In addition, expression of the ZK637.6 transcript was strongly dependent on the expression of miR-249. These findings may lead to a better understanding of miRNA-mediated gene regulation in nematodes., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

RUNX proteins are evolutionarily conserved transcription factors known to be involved in various developmental processes. Here we report a new role for a RUNX protein: a role in stress response. We show that RNT-1, the Caenorhabditis elegans RUNX homolog, is constantly produced and degraded by the ubiquitination-proteasome pathway in the intestine of the nematode. RNT-1 was rapidly stabilized by oxidative stress, and the rnt-1-mutant animals were more sensitive to oxidative stress, indicating that rapid RNT-1 stabilization is a defense response against the oxidative stress. The MAP kinase pathway is required for RNT-1 stabilization, and RNT-1 was phosphorylated by SEK-1/PMK-1 in vitro. ChIP-sequencing analysis revealed a feedback loop mechanism of the MAP kinase pathway by the VHP-1 phosphatase in the RNT-1-mediated oxidative stress response. We propose that rnt-1 is regulated at the protein level for its role in the immediate response to environmental challenges in the intestine.

SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface., (Copyright © 2011 Elsevier Inc. All rights reserved.)

FRM-1 is a member of the FERM protein superfamily containing a FERM domain, which is a highly conserved protein-protein interaction module found in most eukaryotes. Although FRM-1 is thought to be involved in linking intracellular proteins to membrane proteins, the specific role for FRM-1 remains to be elucidated. In an effort to explore the biological function of FRM-1, we examined the phenotype of frm-1(tm4168) mutant worms. We observed that frm-1(tm4168) worms have a delayed hatching phenotype. Twelve hours after being laid, when virtually all wild-type eggs had hatched, only 64% of frm-1(tm4168) eggs had hatched. About 3% of frm-1(tm4168) eggs failed to hatch, even 3 days after they had been laid. We also found that frm-1(tm4168) mutants displayed a temperature-sensitive sterility phenotype. About 13% of frm-1(tm4168) worms were unable to produce eggs or produced nonviable eggs at 25°C. In contrast, less than 1% of wild-type animals were sterile at this temperature. At 20°C, neither the mutant nor wild type appeared to be sterile. Western blot analysis indicates that FRM-1 is expressed throughout the developmental stages with the strongest expression at the egg stage. Immunostaining experiments revealed that FRM-1 is mainly localized to the plasma membrane of most if not all cells at an early embryonic stage and to the plasma membrane of P cells during the late embryonic stages. GFP fusion experiments showed that FRM-1 can be expressed in the pharynx and intestine at the larval and adult stages. Our data suggest that FRM-1 may participate in diverse biological processes, including embryonic development.

RNA interference (RNAi) is a commonly used technique for reverse genetic approaches in Caenorhabditis elegans. Feeding RNAi is the most convenient and inexpensive method for performing genome-wide RNAi screens. However, it has been reported that knock-down of two genes (double RNAi) by feeding RNAi using a mixture of bacteria that each contained one dsRNA species produced poor results. To overcome this problem of inefficiency, we designed and tested a double feeding RNAi method using a single RNAi construct containing two gene fragments. From experiments with three different sets of genes, we found that the new double RNAi method consistently produced significantly enhanced double knock-down phenotypes. The double feeding RNAi approach described here provides a method to consistently examine phenotypes caused by depletion of more than one gene in C. elegans.

The roles of Lats kinases in the regulation of cell proliferation and apoptosis have been well established. Here we report new roles for Lats kinase in the integrity of the apical membrane structure. WTS-1, the C. elegans Lats homolog, localized primarily to the subapical region in the intestine. A loss-of-function mutation in wts-1 resulted in an early larval arrest and defects in the structure of the intestinal lumen. An electron microscopy study of terminally arrested wts-1 mutant animals revealed numerous microvilli-containing lumen-like structures within the intestinal cells. The wts-1 phenotype was not caused by cell proliferation or apoptosis defects. Instead, we found that the wts-1 mutant animals exhibited gradual mislocalization of apical actin and apical junction proteins, suggesting that wts-1 normally suppresses the formation of extra apical membrane structures. Heat-shock-driven pulse-chase expression experiments showed that WTS-1 regulates the localization of newly synthesized apical actins. RNAi of the exocyst complex genes suppressed the mislocalization phenotype of wts-1 mutation. Collectively, the data presented here suggest that Lats kinase plays important roles in the integrity of the apical membrane structure of intestinal cells.

The endoplasmic reticulum (ER) is an organelle associated with lipid metabolism. However, the involvement of the ER in nutritional status-dependent energy homeostasis is largely unknown. We demonstrate that IRE-1, an ER protein known to be involved in the unfolded protein response, and HSP-4, an ER chaperone, regulate expression of the novel fasting-induced lipases FIL-1 and FIL-2, which induce fat granule hydrolysis upon fasting in C. elegans. RNAi and ectopic expression experiments demonstrated that FIL-1 and FIL-2 are both necessary and sufficient for fasting-induced fat granule breakdown. Failure of ire-1 and hsp-4 mutant animals to hydrolyze fat granules during starvation impaired their motility, which was rescued by glucose supplementation, implicating the importance of ire-1/hsp-4-dependent lipolysis for energy supply from stored fat during fasting. These data suggest that the ER-resident proteins IRE-1 and HSP-4 are key nutritional sensors that modulate expression of inducible lipases to maintain whole-body energy homeostasis in C. elegans.

Rapsyn is a postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. Here we report the mechanism for posttranslational control of rapsyn protein stability. We confirmed that C18H9.7-encoded RPY-1 is a rapsyn homolog in Caenorhabditis elegans by showing that human rapsyn rescued rpy-1 mutant phenotypes in nematodes, as determined by levamisole assays and micropost array behavioral assays. We found that RPY-1 was degraded in the absence of functional UNC-29, a non-alpha subunit of the receptor, in an allele-specific manner, but not in the absence of other receptor subunits. The cytoplasmic loop of UNC-29 was found to be critical for RPY-1 stability. Through RNA interference screening, we found that UBC-1, UBC-12, NEDD-8, and RBX-1 were required for degradation of RPY-1. We identified cullin (CUL)-3 as a component of E3 ligase and KEL-8 as the substrate adaptor of RPY-1. Mammalian rapsyn was ubiquitinated by the CUL3/KLHL8-containing E3 ligase in vitro, and the knockdown of KLHL-8, a mammalian KEL-8 homolog, inhibited rapsyn ubiquitination in vivo, implying evolutionary conservation of the rapsyn stability control machinery. kel-8 suppression and rpy-1 overexpression in C. elegans produced a phenotype similar to that of a loss-of-function mutation of rpy-1, suggesting that control of rapsyn abundance is important for proper function of the receptor. Our results suggest a link between the control of rapsyn abundance and congenital myasthenic syndromes.

During development of Caenorhabditis elegans, expression of the RUNX homolog, rnt-1, is tightly regulated both spatially and temporally. In this study, we investigated the mechanism underlying the temporal regulation of rnt-1. We found that rnt-1 contained evolutionarily conserved consensus RUNX binding sequences within one of its introns, and that RNT-1 bound to these intronic sequences both in vitro and in vivo in the presence of BRO-1, suggesting that RNT-1 together with BRO-1 represses its own transcription. Fine deletion and substitution experiments revealed a binding site within the intron that was critical for rnt-1 regulation. Importantly, we found that the TGFbeta homolog, DBL-1, was required for counteracting the repressive activity of BRO-1 at postembryonic stages. Accordingly, ectopic expression of DBL-1 induced transcription of rnt-1 in the lateral hypodermis and other tissues even at the postembryonic stages. Taken together, our data suggest that rnt-1 expression is regulated by the balance between DBL-1-mediated activation and BRO-1-mediated repression at the postembryonic stages.

Tyrosine O-sulfation is one of the post-translational modification processes that occur to membrane proteins and secreted proteins in eukaryotes. Tyrosylprotein sulfotransferase (TPST) is responsible for this modification, and in this report, we describe the expression pattern and the biological role of TPST-A in the nematode Caenorhabditis elegans. We found that TPST-A was mainly expressed in the hypodermis, especially in the seam cells. Reduction of TPST-A activity by RNAi caused severe defects in cuticle formation, indicating that TPST-A is involved in the cuticle formation in the nematode. We found that RNAi of TPST-A suppressed the roller phenotype caused by mutations in the rol-6 collagen gene, suggesting that sulfation of collagen proteins may be important for proper organization of the extracellular cuticle matrix. The TPST-A RNAi significantly decreased the dityrosine level in the worms, raising the possibility that the sulfation process may be a pre-requisite for the collagen tyrosine cross-linking.

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-beta (TGF-beta) signal mediators. However, the involvement of RUNX in TGF-beta signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-beta signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.

Many protein components of telomeres, the multifunctional DNA-protein complexes at the ends of eukaryotic chromosomes, have been identified in diverse species ranging from yeast to humans. In Caenorhabditis elegans, CEH-37 has been identified by a yeast one hybrid screen to be a double-stranded telomere-binding protein. However, the role of CEH-37 in telomere function is unclear because a deletion mutation in this gene does not cause severe telomere defects. This observation raises the possibility of the presence of genetic redundancy. To identify additional double-stranded telomere-binding proteins in C. elegans, we used a different approach, namely, a proteomic approach. Affinity chromatography followed by Finnigan LCQ ion trap mass spectrometer analysis allowed us to identify several candidate proteins. We further characterized one of these, HMG-5, which is encoded by F45E4.9. HMG-5 bound to double-stranded telomere in vitro as shown by competition assays. At least two telomeric DNA repeats were needed for this binding. HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells, but not in the hatched larvae or adults. HMG-5 mainly localized to the chromosomal ends, indicating that HMG-5 also binds to telomeres in vivo. These observations suggest that HMG-5 may participate, together with CEH-37, in early embryogenesis by acting at the telomeres.

In order to characterize the mechanisms regulating neuronal expression of the nematode SNAP-25 gene, we identified the SNAP-25 genes of Caenorhabditis elegans and Caenorhabditis briggsae. Comparative sequence analysis and reporter assays revealed two putative 5' regulatory elements, P1 and P2, and four elements, I1h, I1m, I2h, and I2m, in the first intron. Nuclear extracts contained activities that bound the P2 and I1h elements. Different elements were required for SNAP-25 expression in different neuronal subsets; P1 was required in DA and DD motor neurons, and I1m and I2m were required in DB and DA neurons, respectively. P2 was active in amphid and phasmid neurons, I1h in pharyngeal neurons, and I2h in touch receptor neurons. The I2h element contained a putative binding site for transcription factor UNC-86. Both UNC-86 and MEC-3 were required for I2h activity in the mechanosensory neurons: in these neurons, GFP expression driven by I2h was abolished in animals bearing either an unc-86 null or a mec-3 null mutation, or an unc-86 mutation that leads to defective interaction with MEC-3. Deletion of the MEC-3 binding site also abolished the GFP expression. Gel mobility-shift assay results suggest that transcriptional regulation of SNAP-25 may involve multiple transcription factors.

Caenorhabditis elegans can serve as a model system to study telomere functions due to its similarity to higher organisms in telomere structures. We report here the identification of the nematode homeodomain protein CEH-37 as a telomere-binding protein using a yeast one-hybrid screen. The predicted three-dimensional model of the homeodomain of CEH-37, which has a typical helix-loop-helix structure, was similar to that of the Myb domain of known telomere-binding proteins, which is also a helix-loop-helix protein, despite little amino acid sequence similarity. We demonstrated the specific binding of CEH-37 to the nematode telomere sequences in vitro by competition assays. We determined that CEH-37 binding required at least 1.5 repeats of TTAGGC and that the core sequence for binding was GGCTTA. We found that CEH-37 had an ability to bend telomere sequence-containing DNA, which is the case for other known telomere-binding proteins such as TRF1 and RAP1, indicating that CEH-37 may be involved in establishing or maintaining a secondary structure of the telomeres in vivo. We also demonstrated that CEH-37 was primarily co-localized to the chromosome ends in vivo, indicating that CEH-37 may play roles in telomere functions. Consistent with this, a ceh-37 mutation resulting in a truncated protein caused a weak high incidence of male phenotype, which may have been caused by chromosome instability. The identification of CEH-37 as a telomere-binding protein may represent an evolutionary conservation of telomere-binding proteins in terms of tertiary protein structure rather than primary amino acid sequence.

In this report we have examined two new heat shock protein (HSP16) proteins in the nematode Caenorhabditis elegans encoded by the open reading frames F08H9.3 and F08H9.4. The F08H9.3 and F08H9.4 genes are oriented in the same direction next to each other on the chromosome, not sharing any promoter region, unlike other hsp16 genes that share common promoters in pairs. The F08H9.3 and F08H9.4 proteins were expressed in a tissue-specific manner, unlike the other four HSP16 proteins. F08H9.3 was expressed in the pharynx, and F08H9.4 in the excretory canal and a few neuronal cells. While F08H9.3 was weakly induced by heat shock only in the same tissue as under the normal condition, F08H9.4 was newly induced in the intestine. RNA interference experiments showed that these two proteins are required for survival under the heat shock condition.

The Caenorhabditis elegans run gene encodes a Runt domain factor. Runx1, Runx2, and Runx3 are the three known mammalian homologs of run. Runx1, which plays an essential role in hematopoiesis, has been identified at the breakpoint of chromosome translocations that are responsible for human leukemia. Runx2 plays an essential role in osteogenesis, and inactivation of one allele of Runx2 is responsible for the human disease cleidocranial dysplasia. To understand the role of run in C. elegans, we used transgenic run::GFP reporter constructs and a double-stranded RNA-mediated interference method. The expression of run was detected as early as the bean stage exclusively in the nuclei of seam hypodermal cells and lasted until the L3 stage. At the larval stage, expression of run was additionally detected in intestinal cells. The regulatory elements responsible for the postembryonic hypodermal seam cells and intestinal cells were separately located within a 7.2-kb-long intron region. This is the first report demonstrating that an intron region is essential for stage-specific and cell type-specific expression of a C. elegans gene. RNA interference analysis targeting the run gene resulted in an early larva-lethal phenotype, with apparent malformation of the hypodermis and intestine. These results suggest that run is involved in the development of a functional hypodermis and gut in C. elegans. The highly conserved role of the Runt domain transcription factor in gut development during evolution from nematodes to mammals is discussed.

Telomeres are involved in the maintenance of chromosomes and the prevention of genome instability. Despite this central importance, significant variation in telomere length has been observed in a variety of organisms. The genetic determinants of telomere-length variation and their effects on organismal fitness are largely unexplored. Here, we describe natural variation in telomere length across the Caenorhabditis elegans species. We identify a large-effect variant that contributes to differences in telomere length. The variant alters the conserved oligonucleotide/oligosaccharide-binding fold of protection of telomeres 2 (POT-2), a homolog of a human telomere-capping shelterin complex subunit. Mutations within this domain likely reduce the ability of POT-2 to bind telomeric DNA, thereby increasing telomere length. We find that telomere-length variation does not correlate with offspring production or longevity in C. elegans wild isolates, suggesting that naturally long telomeres play a limited role in modifying fitness phenotypes in C. elegans.