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PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Although the molecular composition and architecture of synapses have been widely explored, much less is known about what genetic programs directly activate synaptic gene expression and how they are modulated. Here, using Caenorhabditis elegans dopaminergic neurons, we reveal that EGL-43/MECOM and FOS-1/FOS control an activity-dependent synaptogenesis program. Loss of either factor severely reduces presynaptic protein expression. Both factors bind directly to promoters of synaptic genes and act together with CUT homeobox transcription factors to activate transcription. egl-43 and fos-1 mutually promote each others expression, and increasing the binding affinity of FOS-1 to the egl-43 locus results in increased presynaptic protein expression and synaptic function. EGL-43 regulates the expression of multiple transcription factors, including activity-regulated factors and developmental factors that define multiple aspects of dopaminergic identity. Together, we describe a robust genetic program underlying activity-regulated synapse formation during development.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the Caenorhabditis elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene lin-66 mediates eif-3.g(gf)-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.

Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated MELT motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

The central nervous system coordinates peripheral cellular stress responses, including the unfolded protein response of the mitochondria (UPRMT); however, the contexts for which this regulatory capability evolved are unknown. UPRMT is up-regulated upon pathogenic infection and in metabolic flux, and the olfactory nervous system has been shown to regulate pathogen resistance and peripheral metabolic activity. Therefore, we asked whether the olfactory nervous system in Caenorhabditis elegans controls the UPRMT cell nonautonomously. We found that silencing a single inhibitory olfactory neuron pair, AWC, led to robust induction of UPRMT and reduction of oxidative phosphorylation dependent on serotonin signaling and parkin-mediated mitophagy. Further, AWC ablation confers resistance to the pathogenic bacteria Pseudomonas aeruginosa partially dependent on the UPRMT transcription factor atfs-1 and fully dependent on mitophagy machinery. These data illustrate a role for the olfactory nervous system in regulating whole-organism mitochondrial dynamics, perhaps in preparation for postprandial metabolic stress or pathogenic infection.

Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

In spliceosome assembly, the 5 splice site is initially recognized by U1 snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to the maintenance of 5 splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in Caenorhabditis elegans at SNRP-27 M141 promote changes in alternative 5ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5 splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5 splice site usage. Many of the alternative 5 splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load alternative 5 splice sites into the active site. Tests with mutant U1 snRNA and swapped 5 splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5 splice alternative splicing event is dependent on both the presence of a weaker consensus 5ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C terminus of SNU-66 in maintenance of 5 splice site identity during spliceosome assembly.

Insulin-mechanistic target of rapamycin (mTOR) signaling drives anabolic growth during organismal development; its late-life dysregulation contributes to aging and limits lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here, we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin, INS-7, is drastically overproduced from early life and shortens lifespan in lpd-3 mutants. LPD-3 forms a bridge-like tunnel megaprotein to facilitate non-vesicular cellular lipid trafficking. Lipidomic profiling reveals increased hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1. Reducing the abundance of HYL-1, insulin receptor/DAF-2 or mTOR/LET-363, normalizes INS-7 levels and rescues the lifespan of lpd-3 mutants. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age. We propose that LPD-3 acts as a megaprotein brake for organismal aging and that its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

An animals preference for many chemosensory cues remains constant despite dramatic changes in the animals internal state. The mechanisms that maintain chemosensory preference across different physiological contexts remain poorly understood. We previously showed that distinct patterns of neural activity and motor output are evoked by carbon dioxide (CO2) in starved adults vs dauers of Caenorhabditis elegans, despite the two life stages displaying the same preference (attraction) for CO2. However, how the distinct CO2-evoked neural dynamics and motor patterns contribute to CO2 attraction at the two life stages remained unclear. Here, using a CO2 chemotaxis assay, we show that different interneurons are employed to drive CO2 attraction at the two life stages. We also investigate the molecular mechanisms that mediate CO2 attraction in dauers vs adults. We show that insulin signaling promotes CO2 attraction in dauers but not starved adults and that different combinations of neurotransmitters and neuropeptides are used for CO2 attraction at the two life stages. Our findings provide new insight into the distinct molecular and cellular mechanisms used by C. elegans at two different life stages to generate attractive behavioral responses to CO2.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. Increased TSP-1 abundance persists even after transient early-life heat stress. Such regulation requires CBP-1, a histone acetyltransferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity.

Progranulin is an evolutionarily conserved protein that has been implicated in human neurodevelopmental and neurodegenerative diseases. Human progranulin is comprised of multiple cysteine-rich, biologically active granulin peptides. Granulin peptides accumulate with age and stress, however their functional contributions relative to full-length progranulin remain unclear. To address this, we generated C. elegans strains that produced quantifiable levels of both full-length progranulin/PGRN-1 protein and cleaved granulin peptide. Using these strains, we demonstrated that even in the presence of intact PGRN-1, granulin peptides suppressed the activity of the lysosomal aspartyl protease activity, ASP-3/CTSD. Granulin peptides were also dominant over PGRN-1 in compromising animal fitness as measured by progress through development and stress response. Finally, the degradation of human TDP-43 was impaired when the granulin to PGRN-1 ratio was increased, representing a disease-relevant downstream impact of impaired lysosomal function. In summary, these studies suggest that not only absolute progranulin levels, but also the balance between full-length progranulin and its cleavage products, is important in regulating lysosomal biology. Given its relevance in human disease, this suggests that the processing of progranulin into granulins should be considered as part of disease pathobiology and may represent a site of therapeutic intervention.

Dysfunctional extracellular matrices (ECM) contribute to aging and disease. Repairing dysfunctional ECM could potentially prevent age-related pathologies. Interventions promoting longevity also impact ECM gene expression. However, the role of ECM composition changes in healthy aging remains unclear. Here we perform proteomics and in-vivo monitoring to systematically investigate ECM composition (matreotype) during aging in C. elegans revealing three distinct collagen dynamics. Longevity interventions slow age-related collagen stiffening and prolong the expression of collagens that are turned over. These prolonged collagen dynamics are mediated by a mechanical feedback loop of hemidesmosome-containing structures that span from the exoskeletal ECM through the hypodermis, basement membrane ECM, to the muscles, coupling mechanical forces to adjust ECM gene expression and longevity via the transcriptional co-activator YAP-1 across tissues. Our results provide in-vivo evidence that coordinated ECM remodeling through mechanotransduction is required and sufficient to promote longevity, offering potential avenues for interventions targeting ECM dynamics.

Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer (blistering, Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale.

Although the type-I interferon (IFN-I) response is considered vertebrate-specific, recent findings about the Intracellular Pathogen Response (IPR) in nematode Caenorhabditis elegans indicate that there are similarities between these two transcriptional immunological programs. The IPR is induced during infection with natural intracellular fungal and viral pathogens of the intestine and promotes resistance against these pathogens. Similarly, the IFN-I response is induced by viruses and other intracellular pathogens and promotes resistance against infection. Whether the IPR and the IFN-I response evolved in a divergent or convergent manner is an unanswered and exciting question, which could be addressed by further studies of immunity against intracellular pathogens in C. elegans and other simple host organisms. Here we highlight similar roles played by RIG-I-like receptors, purine metabolism enzymes, proteotoxic stressors, and transcription factors to induce the IPR and IFN-I response, as well as the similar consequences of these defense programs on organismal development.

The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). Purifying selection mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5, which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNAuaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNAuaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.

Primary cilia are specialized organelles supporting the development and function of cells and organisms. Intraflagellar transport (IFT) is essential for cilia formation, maintenance, and function. In C. elegans ciliated sensory neurons, IFT interacts with signaling molecules to generate distinct morphological and function features and also to maintain the integrity of cilia. Here, we report an IFT-dependent feedback control on the conserved MAPKKK DLK-1 in the ciliated sensory neurons. DLK proteins are widely known to act in synapse formation, axon regeneration, and degeneration, but their roles in other neuronal compartments are understudied. By forward genetic screening for altered expression of the endogenously tagged DLK-1 we identified multiple ift mutants showing increased DLK-1 accumulation in the defective sensory endings. We show that in response to acute IFT disruption, DLK-1 accumulates rapidly and reversibly. The expression levels of the transcription factor CEBP-1, known to act downstream of DLK-1 in the development and maintenance of synapses and axons, are also increased in the ciliated sensory neurons of ift mutants. Interestingly, the regulation of CEBP-1 expression shows sensory neuron-type dependency on DLK-1. Moreover, in the sensory neuron AWC, which has elaborate cilia morphology, up-regulated CEBP-1 represses DLK-1 at the transcription level, thereby dampening DLK-1 accumulation. Last, the IFT-dependent regulatory loop of DLK-1 and CEBP-1 offers neuroprotection in a cilia degeneration model. These findings uncover a surveillance mechanism in which tight control on the DLK-1 signaling protects cilia integrity in a context-specific manner.

The Maillard reaction, a chemical reaction between amino acids and sugars, is exploited to produce flavorful food ubiquitously, from the baking industry to our everyday lives. However, the Maillard reaction also occurs in all cells, from prokaryotes to eukaryotes, forming advanced glycation end-products (AGEs). AGEs are a heterogeneous group of compounds resulting from the irreversible reaction between biomolecules and α-dicarbonyls (α-DCs), including methylglyoxal (MGO), an unavoidable byproduct of anaerobic glycolysis and lipid peroxidation. We previously demonstrated that Caenorhabditis elegans mutants lacking the glod-4 glyoxalase enzyme displayed enhanced accumulation of α-DCs, reduced lifespan, increased neuronal damage, and touch hypersensitivity. Here, we demonstrate that glod-4 mutation increased food intake and identify that MGO-derived hydroimidazolone, MG-H1, is a mediator of the observed increase in food intake. RNAseq analysis in glod-4 knockdown worms identified upregulation of several neurotransmitters and feeding genes. Suppressor screening of the overfeeding phenotype identified the tdc-1-tyramine-tyra-2/ser-2 signaling as an essential pathway mediating AGE (MG-H1)-induced feeding in glod-4 mutants. We also identified the elt-3 GATA transcription factor as an essential upstream regulator for increased feeding upon accumulation of AGEs by partially controlling the expression of tdc-1 gene. Furthermore, the lack of either tdc-1 or tyra-2/ser-2 receptors suppresses the reduced lifespan and rescues neuronal damage observed in glod-4 mutants. Thus, in C. elegans, we identified an elt-3 regulated tyramine-dependent pathway mediating the toxic effects of MG-H1 AGE. Understanding this signaling pathway may help understand hedonistic overfeeding behavior observed due to modern AGE-rich diets.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans-the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity, and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside and at the apical side of intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans.

Gene activity defines cell identity, drives intercellular communication, and underlies the functioning of multicellular organisms. We present the single-cell resolution atlas of gene activity of a fertile adult metazoan: Caenorhabditis elegans. This compendium comprises 180 distinct cell types and 19,657 expressed genes. We predict 7541 transcription factor expression profile associations likely responsible for defining cellular identity. We predict thousands of intercellular interactions across the C. elegans body and the ligand-receptor pairs that mediate them, some of which we experimentally validate. We identify 172 genes that show consistent expression across cell types, are involved in basic and essential functions, and are conserved across phyla; therefore, we present them as experimentally validated housekeeping genes. We developed the WormSeq application to explore these data. In addition to the integrated gene-to-systems biology, we present genome-scale single-cell resolution testable hypotheses that we anticipate will advance our understanding of the molecular mechanisms, underlying the functioning of a multicellular organism and the perturbations that lead to its malfunction.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Many chemosensory cues evoke responses of the same valence under widely varying physiological conditions. It remains unclear whether similar or distinct neural mechanisms are involved in the detection and processing of such chemosensory cues across contexts. We show that in Caenorhabditis elegans, a chemosensory cue is processed by distinct neural mechanisms at two different life stages that share the same valence state. Both starved adults and dauer larvae are attracted to carbon dioxide (CO2), but CO2 evokes different patterns of neural activity and different motor outputs at the two life stages. Moreover, the same interneuron within the CO2 microcircuit plays a different role in driving CO2-evoked motor output at the two life stages. The dauer-specific patterns of CO2-evoked activity in this interneuron require a dauer-specific gap junction complex and insulin signaling. Our results demonstrate that functionally distinct microcircuits are engaged in response to a chemosensory cue that triggers the same valence state at different life stages, revealing an unexpected complexity to chemosensory processing.

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.

Chromosome segregation errors during meiosis are the leading cause of aneuploidy. Faithful chromosome segregation during meiosis in most eukaryotes requires a crossover which provides a physical attachment holding homologs together in a "bivalent." Crossovers are critical for homologs to be properly aligned and partitioned in the first meiotic division. Without a crossover, individual homologs (univalents) might segregate randomly, resulting in aneuploid progeny. However, Caenorhabditis elegans zim-2 mutants, which have crossover defects on chromosome V, have fewer dead embryos than that expected from random segregation. This deviation from random segregation is more pronounced in zim-2 males than that in females. We found three phenomena that can explain this apparent discrepancy. First, we detected crossovers on chromosome V in both zim-2(tm574) oocytes and spermatocytes, suggesting a redundant mechanism to make up for the ZIM-2 loss. Second, after accounting for the background crossover frequency, spermatocytes produced significantly more euploid gametes than what would be expected from random segregation. Lastly, trisomy of chromosome V is viable and fertile. Together, these three phenomena allow zim-2(tm574) mutants with reduced crossovers on chromosome V to have more viable progeny. Furthermore, live imaging of meiosis in spo-11(me44) oocytes and spermatocytes, which exhibit crossover failure on all 6 chromosomes, showed 12 univalents segregating apart in roughly equal masses in a homology-independent manner, supporting the existence of a mechanism that segregates any 2 chromosomes apart.

The nematode Caenorhabditis elegans is a research model organism particularly suited to the mechanistic understanding of synapse genesis in the nervous system. Armed with powerful genetics, knowledge of complete connectomics, and modern genomics, studies using C. elegans have unveiled multiple key regulators in the formation of a functional synapse. Importantly, many signaling networks display remarkable conservation throughout animals, underscoring the contributions of C. elegans research to advance the understanding of our brain. In this chapter, we will review up-to-date information of the contribution of C. elegans to the understanding of chemical synapses, from structure to molecules and to synaptic remodeling.

BackgroundPeriodic prolonged fasting (PF) extends lifespan in model organisms and ameliorates multiple disease states both clinically and experimentally owing, in part, to its ability to modulate the immune system. However, the relationship between metabolic factors, immunity, and longevity during PF remains poorly characterized especially in humans.ObjectiveThis study aimed to observe the effects of PF in human subjects on the clinical and experimental markers of metabolic and immune health and uncover underlying plasma-borne factors that may be responsible for these effects.MethodsIn this rigorously controlled pilot study (ClinicalTrial.gov identifier, NCT03487679), 20 young males and females participated in a 3-d study protocol including assessments of 4 distinct metabolic states: 1) overnight fasted baseline state, 2) 2-h postprandial fed state, 3) 36-h fasted state, and 4) final 2-h postprandial re-fed state 12 h after the 36-h fasting period. Clinical and experimental markers of immune and metabolic health were assessed for each state along with comprehensive metabolomic profiling of participant plasma. Bioactive metabolites identified to be upregulated in circulation after 36 h of fasting were then assessed for their ability to mimic the effects of fasting in isolated human macrophage as well as the ability to extend lifespan in Caenorhabditis elegans.ResultsWe showed that PF robustly altered the plasma metabolome and conferred beneficial immunomodulatory effects on human macrophages. We also identified 4 bioactive metabolites that were upregulated during PF (spermidine, 1-methylnicotinamide, palmitoylethanolamide, and oleoylethanolamide) that could replicate these immunomodulatory effects. Furthermore, we found that these metabolites and their combination significantly extended the median lifespan of C. elegans by as much as 96%.ConclusionsThe results of this study reveal multiple functionalities and immunological pathways affected by PF in humans, identify candidates for the development of fasting mimetic compounds, and uncover targets for investigation in longevity research.

Oocyte meiotic spindles mediate the expulsion of ¾ of the genome into polar bodies to generate diploid zygotes in nearly all animal species. Failures in this process result in aneuploid or polyploid offspring that are typically inviable. Accurate meiotic chromosome segregation and polar body extrusion require the spindle to elongate while maintaining its structural integrity. Previous studies have implicated three hypothetical activities during this process, including microtubule crosslinking, microtubule sliding and microtubule polymerization. However, how these activities regulate spindle rigidity and elongation as well as the exact proteins involved in the activities remain unclear. We discovered that C. elegans meiotic anaphase spindle integrity is maintained through redundant microtubule crosslinking activities of the Kinesin-5 family motor BMK-1, the microtubule bundling protein SPD-1/PRC1, and the Kinesin-4 family motor, KLP-19. Using time-lapse imaging, we found that single depletion of KLP-19KIF4A, SPD-1PRC1 or BMK-1Eg5 had minimal effects on anaphase B spindle elongation velocity. In contrast, double depletion of SPD-1PRC1 and BMK-1Eg5 or double depletion of KLP-19KIF4A and BMK-1Eg5 resulted in spindles that elongated faster, bent in a myosin-dependent manner, and had a high rate of polar body extrusion errors. Bending spindles frequently extruded both sets of segregating chromosomes into two separate polar bodies. Normal anaphase B velocity was observed after double depletion of KLP-19KIF4A and SPD-1PRC1. These results suggest that KLP-19KIF4A and SPD-1PRC1 act in different pathways, each redundant with a separate BMK-1Eg5 pathway in regulating meiotic spindle elongation. Depletion of ZYG-8, a doublecortin-related microtubule binding protein, led to slower anaphase B spindle elongation. We found that ZYG-8DCLK1 acts by excluding SPD-1PRC1 from the spindle. Thus, three mechanistically distinct microtubule regulation modules, two based on crosslinking, and one based on exclusion of crosslinkers, power the mechanism that drives spindle elongation and structural integrity during anaphase B of C.elegans female meiosis.

How an organism dies is a fundamental yet poorly understood question in biology. An organism can die of many causes, including stress-induced phenoptosis, also defined as organismic death that is regulated by its genome-encoded programs. The mechanism of stress-induced phenoptosis is still largely unknown. Here, we show that transient but severe freezing-thaw stress (FTS) in Caenorhabditis elegans induces rapid and robust phenoptosis that is regulated by G-protein coupled receptor (GPCR) signaling. RNAi screens identify the GPCR-encoding fshr-1 in mediating transcriptional responses to FTS. FSHR-1 increases ligand interaction upon FTS and activates a cyclic AMP-PKA cascade leading to a genetic program to promote organismic death under severe stress. FSHR-1/GPCR signaling up-regulates the bZIP-type transcription factor ZIP-10, linking FTS to expression of genes involved in lipid remodeling, proteostasis, and aging. A mathematical model suggests how genes may promote organismic death under severe stress conditions, potentially benefiting growth of the clonal population with individuals less stressed and more reproductively privileged. Our studies reveal the roles of FSHR-1/GPCR-mediated signaling in stress-induced gene expression and phenoptosis in C. elegans, providing empirical new insights into mechanisms of stress-induced phenoptosis with evolutionary implications.

Amygdalin is a cyanogenic glycoside enriched in the tissues of many edible plants, including seeds of stone fruits such as cherry (Prunus avium), peach (Prunus persica), and apple (Malus domestica). These plants biosynthesize amygdalin in defense against herbivore animals, as amygdalin generates poisonous cyanide upon plant tissue destruction.1,2,3,4 Poisonous to many animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals.5 Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1, and cysl-2. A screen of a natural product library for hypoxia-independent regulators of HIF-1 identifies amygdalin as a potent activator of cysl-2, a HIF-1 transcriptional target that encodes a cyanide detoxification enzyme in C. elegans. As a cysl-2 paralog similarly essential for amygdalin resistance, cysl-1 encodes a protein homologous to cysteine biosynthetic enzymes in bacteria and plants but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis indicates that cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Since acquisition, these two genes evolved division of labor in a cellular circuit to detect and detoxify cyanide. Thus, algae-to-nematode HGT and subsequent gene function co-option events may facilitate host survival and adaptation to adverse environmental stresses and biogenic toxins.

The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5 splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5 splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5ss identified by U1snRNA for entry into the catalytic core.

Spermatogenesis is the process through which mature male gametes are formed and is necessary for the transmission of genetic information. While much work has established how sperm fate is promoted and maintained, less is known about how the sperm morphogenesis program is executed. We previously identified a novel role for the nuclear hormone receptor transcription factor, NHR-23, in promoting Caenorhabditis elegans spermatogenesis. The depletion of NHR-23 along with SPE-44, another transcription factor that promotes spermatogenesis, caused additive phenotypes. Through RNA-seq, we determined that NHR-23 and SPE-44 regulate distinct sets of genes. The depletion of both NHR-23 and SPE-44 produced yet another set of differentially regulated genes. NHR-23-regulated genes are enriched in phosphatases, consistent with the switch from genome quiescence to post-translational regulation in spermatids. In the parasitic nematode Ascaris suum, MFP1 and MFP2 control the polymerization of Major Sperm Protein, the molecule that drives sperm motility and serves as a signal to promote ovulation. NHR-23 and SPE-44 regulate several MFP2 paralogs, and NHR-23 depletion from the male germline caused defective localization of MSD/MFP1 and NSPH-2/MFP2. Although NHR-23 and SPE-44 do not transcriptionally regulate the casein kinase gene spe-6, a key regulator of sperm development, SPE-6 protein is lost following NHR-23+SPE-44 depletion. Together, these experiments provide the first mechanistic insight into how NHR-23 promotes spermatogenesis and an entry point to understanding the synthetic genetic interaction between nhr-23 and spe-44.

Lysosomes are key cellular organelles that metabolize extra- and intracellular substrates. Alterations in lysosomal metabolism are implicated in ageing-associated metabolic and neurodegenerative diseases. However, how lysosomal metabolism actively coordinates the metabolic and nervous systems to regulate ageing remains unclear. Here we report a fat-to-neuron lipid signalling pathway induced by lysosomal metabolism and its longevity-promoting role in Caenorhabditis elegans. We discovered that induced lysosomal lipolysis in peripheral fat storage tissue upregulates the neuropeptide signalling pathway in the nervous system to promote longevity. This cell-non-autonomous regulation is mediated by a specific polyunsaturated fatty acid, dihomo-γ-linolenic acid, and LBP-3 lipid chaperone protein transported from the fat storage tissue to neurons. LBP-3 binds to dihomo-γ-linolenic acid, and acts through NHR-49 nuclear receptor and NLP-11 neuropeptide in neurons to extend lifespan. These results reveal lysosomes as a signalling hub to coordinate metabolism and ageing, and lysosomal signalling mediated inter-tissue communication in promoting longevity.

During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules.1,2 One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin.3 Here, we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-CHCP-4 act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-CHCP-4 resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore.4,5 This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans: centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.

Biological sex affects numerous aspects of biology, yet how sex influences different biological processes have not been extensively studied at the molecular level. Caenorhabditis elegans, with both hermaphrodites (functionally females as adults) and males, is an excellent system to uncover how sex influences physiology. Here, we describe a method to isolate large quantities of C. elegans males by conditionally degrading DPY-27, a component of the dosage compensation complex essential for hermaphrodite, but not male, development. We show that germ cells from males isolated following DPY-27 degradation undergo meiosis and spermiogenesis like wild type and these males are competent to mate and sire viable offspring. We further demonstrate the efficacy of this system by analyzing gene expression and performing affinity pull-downs from male worm extracts.

microRNAs (miRNAs) are crucial for normal development and physiology. To identify factors that might coordinate with miRNAs to regulate gene expression, we used 2O-methylated oligonucleotides to precipitate Caenorhabditis elegans let-7, miR-58, and miR-2 miRNAs and the associated proteins. A total of 211 proteins were identified through mass-spectrometry analysis of miRNA co-precipitates, which included previously identified interactors of key miRNA pathway components. Gene ontology analysis of the identified interactors revealed an enrichment for RNA binding proteins, suggesting that we captured proteins that may be involved in mRNA lifecycle. To determine which miRNA interactors are important for miRNA activity, we used RNAi to deplete putative miRNA co-factors in animals with compromised miRNA activity and looked for alterations of the miRNA mutant phenotypes. Depletion of 25 of 39 tested genes modified the miRNA mutant phenotypes in three sensitized backgrounds. Modulators of miRNA phenotypes ranged from RNA binding proteins RBD-1 and CEY-1 to metabolic factors such as DLST-1 and ECH-5, among others. The observed functional interactions suggest widespread coordination of these proteins with miRNAs to ultimately regulate gene expression. This study provides a foundation for future investigations aimed at deciphering the molecular mechanisms of miRNA-mediated gene regulation.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.

In this issue of PLOS Biology, Lattmann and colleagues report a new function for proteins of the DNA prereplication complex promoting the anchor cell to invade through the basement membrane and initiate vulval development in Caenorhabditis elegans.

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Coordinating the balance between development and stress responses is critical for organismal survival. However, the cellular signaling controlling this mechanism is not well understood. In Caenorhabditis elegans, it has been hypothesized that a genetic network regulated by NIPI-3/Tibbles may control the balance between animal development and immune response. Using a nipi-3(0) lethality suppressor screen in C. elegans, we reveal a novel role for N-terminal acetyltransferase C complex natc-1/2/3 and histone deacetylase hda-4, in the control of animal development. These signaling proteins act, at least in part, through a PMK-1 p38 MAP kinase pathway (TIR-1-NSY-1-SEK-1-PMK-1), which plays a critical role in the innate immunity against infection. Additionally, using a transcriptional reporter of SEK-1, a signaling molecule within this p38 MAP kinase system that acts directly downstream of C/EBP bZip transcription factor CEBP-1, we find unexpected positive control of sek-1 transcription by SEK-1 along with several other p38 MAP kinase pathway components. Together, these data demonstrate a role for NIPI-3 regulators in animal development, operating, at least in part through a PMK-1 p38 MAPK pathway. Because the C. elegans p38 MAP kinase pathway is well known for its role in cellular stress responses, the novel biological components and mechanisms pertaining to development identified here may also contribute to the balance between stress response and development.

A family of giant KASH proteins, including C. elegans ANC-1 and mammalian Nesprin-1 and -2, are involved in organelle anchoring and are associated with multiple neurodevelopmental disorders including autism, bipolar disorder, and schizophrenia. However, little is known about how these proteins function in neurons. Moreover, the role of organelle anchoring in axon development is poorly understood. Here, we report that ANC-1 functions with the SLT-1 extracellular guidance cue to polarize ALM axon growth. This role for ANC-1 is specific to its longer ANC-1A and ANC-1C isoforms, suggesting that it is mechanistically distinct from previously described roles for ANC-1. We find that ANC-1 is required for the localization of a cluster of mitochondria to the base of the proximal axon. Furthermore, genetic and pharmacological studies indicate that ANC-1 functions with mitochondria to promote polarization of ALM axon growth. These observations reveal a mechanism whereby ANC-1 functions through mitochondria to polarize axon growth in response to SLT-1.

BackgroundOrganisms in the wild can acquire disease- and stress-resistance traits that outstrip the programs endogenous to humans. Finding the molecular basis of such natural resistance characters is a key goal of evolutionary genetics. Standard statistical-genetic methods toward this end can perform poorly in organismal systems that lack high rates of meiotic recombination, like Caenorhabditis worms.ResultsHere we discovered unique ER stress resistance in a wild Kenyan C. elegans isolate, which in inter-strain crosses was passed by hermaphrodite mothers to hybrid offspring. We developed an unbiased version of the reciprocal hemizygosity test, RH-seq, to explore the genetics of this parent-of-origin-dependent phenotype. Among top-scoring gene candidates from a partial-coverage RH-seq screen, we focused on the neuronally-expressed, cuticlin-like gene cutl-24 for validation. In gene-disruption and controlled crossing experiments, we found that cutl-24 was required in Kenyan hermaphrodite mothers for ER stress tolerance in their inter-strain hybrid offspring; cutl-24 was also a contributor to the trait in purebred backgrounds.ConclusionsThese data establish the Kenyan strain allele of cutl-24 as a determinant of a natural stress-resistant state, and they set a precedent for the dissection of natural trait diversity in invertebrate animals without the need for a panel of meiotic recombinants.

Microsporidia are poorly understood, ubiquitous eukaryotic parasites that are completely dependent on their hosts for replication. With the discovery of microsporidia species naturally infecting the genetically tractable transparent nematode C. elegans, this host has been used to explore multiple areas of microsporidia biology. Here we review results about microsporidia infections in C. elegans, which began with the discovery of the intestinal-infecting species Nematocida parisii. Recent findings include new species identification in the Nematocida genus, with more intestinal-infecting species, and also a species with broader tissue tropism, the epidermal and muscle-infecting species Nematocida displodere. This species has a longer polar tube infection apparatus, which may enable its wider tissue range. After invasion, multiple Nematocida species appear to fuse host cells, which likely promotes their dissemination within host organs. Localized proteomics identified Nematocida proteins that have direct contact with the C. elegans intestinal cytosol and nucleus, and many of these host-exposed proteins belong to expanded, species-specific gene families. On the host side, forward genetic screens have identified regulators of the Intracellular Pathogen Response (IPR), which is a transcriptional response induced by both microsporidia and the Orsay virus, which is also a natural, obligate intracellular pathogen of the C. elegans intestine. The IPR constitutes a novel immune/stress response that promotes resistance against microsporidia, virus, and heat shock. Overall, the Nematocida/C. elegans system has provided insights about strategies for microsporidia pathogenesis, as well as innate defense pathways against these parasites.

Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression ofaaim-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favored in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild-type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.