Mechanism of an animal toxin-antidote system.

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

Mutations in the NXF-1:NXT-1 mRNA export complex affect gene-expression driven by the hsp-16.41 promoter.

The NXF-1 : NXT-1 heterodimer is essential for the nuclear export of mRNA. Here we describe three new alleles of nxf-1 and one allele of nxt-1 isolated from a forward genetic screen. These mutations cause no apparent phenotype under normal growth conditions, but partially suppress the lethality caused by heat-shock induced expression of the PEEL-1 toxin from P hsp-16.41 :: peel-1 . There is also decreased expression of P hsp-16.41 ::eGFP in an nxf-1 mutant. We propose that NXF-1 : NXT-1 influences the expression of heat-shock activated genes due to a role in the recruitment of the hsp-16.41 promoter to the nuclear pore complex during heat-shock.

Effectors of anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. At present, the factors that contribute to aECM function are mostly unknown, including the aECM components themselves, their posttranslational regulators, and the pathways required for their secretion. Here we showed that two proteins previously linked to aECM function, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function in a complex. Proteomics experiments also suggested potential roles for SYM-3/FAM102A and SYM-4/WDR44 family proteins in intracellular trafficking. Nonetheless, we found no evidence to support a critical function for SYM-3 or SYM-4 in the apical deposition of two aECM components, NOAH-1 and FBN-1. Moreover, loss of a key splicing regulator of fbn-1, MEC-8/RBPMS2, had surprisingly little effect on the abundance or deposition of FBN-1. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. We also characterized morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a similar phenotype to mec-8; sym double mutants. Collectively, these findings add to our knowledge of factors controlling embryonic morphogenesis.

Pathways that affect anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. We showed that two conserved proteins linked to this process, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function together in a complex. Proteomics data also suggested potential roles for FAM102A and WDR44 family proteins in intracellular trafficking, consistent with their localization patterns. Nonetheless, we found no evidence to support a clear function for SYM-3 or SYM-4 in the apical deposition of two aECM components, FBN-1 and NOAH. Surprisingly, loss of MEC-8/RBPMS2, a conserved splicing factor and regulator of fbn-1 , had little effect on the abundance or deposition of FBN-1 to the aECM. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. Lastly, we examined morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a related embryonic phenotype to mec-8; sym double mutants. Collectively, our findings add to our knowledge of pathways controlling embryonic morphogenesis. SUMMARY STATEMENT: We identify new proteins in apical ECM biology in C. elegans and provide evidence that SYM-3/FAM102A and SYM-4/WDR44 function together in trafficking but do not regulate apical ECM protein deposition.

G protein-coupled receptor kinase-2 (GRK-2) controls exploration through neuropeptide signaling in Caenorhabditis elegans.

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, C. elegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in C. elegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior.

Recurrent but Short-Lived Duplications of Centromeric Proteins in Holocentric Caenorhabditis Species.

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in 32 Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and 10 independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1-2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69-100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential subfunctionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid "revolving door" of kinetochore protein paralogs.

Local adaptation and spatiotemporal patterns of genetic diversity revealed by repeated sampling of Caenorhabditis elegans across the Hawaiian Islands.

The nematode Caenorhabditis elegans is among the most widely studied organisms, but relatively little is known about its natural ecology. Genetic diversity is low across much of the globe but high in the Hawaiian Islands and across the Pacific Rim. To characterize the niche and genetic diversity of C. elegans on the Hawaiian Islands and to explore how genetic diversity might be influenced by local adaptation, we repeatedly sampled nematodes over a three-year period, measured various environmental parameters at each sampling site, and whole-genome sequenced the C. elegans isolates that we identified. We found that the typical Hawaiian C. elegans niche comprises moderately moist native forests at high elevations (500-1,500 m) where ambient air temperatures are cool (15-20°C). Compared to other Caenorhabditis species found on the Hawaiian Islands (e.g., Caenorhabditis briggsae and Caenorhabditis tropicalis), we found that C. elegans were enriched in native habitats. We measured levels of genetic diversity and differentiation among Hawaiian C. elegans and found evidence of seven genetically distinct groups distributed across the islands. Then, we scanned these genomes for signatures of local adaptation and identified 18 distinct regions that overlap with hyper-divergent regions, which may be maintained by balancing selection and are enriched for genes related to environmental sensing, xenobiotic detoxification, and pathogen resistance. These results provide strong evidence of local adaptation among Hawaiian C. elegans and contribute to our understanding of the forces that shape genetic diversity on the most remote volcanic archipelago in the world.

Dopamine receptor DOP-1 engages a sleep pathway to modulate swimming in C. elegans.

Animals require robust yet flexible programs to support locomotion. Here we report a pathway that connects the D1-like dopamine receptor DOP-1 with a sleep mechanism to modulate swimming in C. elegans. We show that DOP-1 plays a negative role in sustaining swimming behavior. By contrast, a pathway through the D2-like dopamine receptor DOP-3 negatively regulates the initiation of swimming, but its impact fades quickly over a few minutes. We find that DOP-1 and the GPCR kinase (G-protein-coupled receptor kinase-2) function in the sleep interneuron RIS, where DOP-1 modulates the secretion of a sleep neuropeptide FLP-11. We further show that DOP-1 and FLP-11 act in the same pathway to modulate swimming. Together, these results delineate a functional connection between a dopamine receptor and a sleep program to regulate swimming in C. elegans. The temporal transition between DOP-3 and DOP-1 pathways highlights the dynamic nature of neuromodulation for rhythmic movements that persist over time.

Casein Kinase 1δ Stabilizes Mature Axons by Inhibiting Transcription Termination of Ankyrin.

After axon outgrowth and synapse formation, the nervous system transitions to a stable architecture. In C. elegans, this transition is marked by the appearance of casein kinase 1δ (CK1δ) in the nucleus. In CK1δ mutants, neurons continue to sprout growth cones into adulthood, leading to a highly ramified nervous system. Nervous system architecture in these mutants is completely restored by suppressor mutations in ten genes involved in transcription termination. CK1δ prevents termination by phosphorylating and inhibiting SSUP-72. SSUP-72 would normally remodel the C-terminal domain of RNA polymerase in anticipation of termination. The antitermination activity of CK1δ establishes the mature state of a neuron by promoting the expression of the long isoform of a single gene, the cytoskeleton protein Ankyrin.

EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells.

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargoes including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting-protein EIPR-1 as proteins important for controlling levels of DCV cargoes in Caenorhabditis elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargoes such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

Hybridization promotes asexual reproduction in Caenorhabditis nematodes.

Although most unicellular organisms reproduce asexually, most multicellular eukaryotes are obligately sexual. This implies that there are strong barriers that prevent the origin or maintenance of asexuality arising from an obligately sexual ancestor. By studying rare asexual animal species we can gain a better understanding of the circumstances that facilitate their evolution from a sexual ancestor. Of the known asexual animal species, many originated by hybridization between two ancestral sexual species. The balance hypothesis predicts that genetic incompatibilities between the divergent genomes in hybrids can modify meiosis and facilitate asexual reproduction, but there are few instances where this has been shown. Here we report that hybridizing two sexual Caenorhabditis nematode species (C. nouraguensis females and C. becei males) alters the normal inheritance of the maternal and paternal genomes during the formation of hybrid zygotes. Most offspring of this interspecies cross die during embryogenesis, exhibiting inheritance of a diploid C. nouraguensis maternal genome and incomplete inheritance of C. becei paternal DNA. However, a small fraction of offspring develop into viable adults that can be either fertile or sterile. Fertile offspring are produced asexually by sperm-dependent parthenogenesis (also called gynogenesis or pseudogamy); these progeny inherit a diploid maternal genome but fail to inherit a paternal genome. Sterile offspring are hybrids that inherit both a diploid maternal genome and a haploid paternal genome. Whole-genome sequencing of individual viable worms shows that diploid maternal inheritance in both fertile and sterile offspring results from an altered meiosis in C. nouraguensis oocytes and the inheritance of two randomly selected homologous chromatids. We hypothesize that hybrid incompatibility between C. nouraguensis and C. becei modifies maternal and paternal genome inheritance and indirectly induces gynogenetic reproduction. This system can be used to dissect the molecular mechanisms by which hybrid incompatibilities can facilitate the emergence of asexual reproduction.

Pristionchus nematodes occur frequently in diverse rotting vegetal substrates and are not exclusively necromenic, while Panagrellus redivivoides is found specifically in rotting fruits.

The lifestyle and feeding habits of nematodes are highly diverse. Several species of Pristionchus (Nematoda: Diplogastridae), including Pristionchus pacificus, have been reported to be necromenic, i.e. to associate with beetles in their dauer diapause stage and wait until the death of their host to resume development and feed on microbes in the decomposing beetle corpse. We review the literature and suggest that the association of Pristionchus to beetles may be phoretic and not necessarily necromenic. The view that Pristionchus nematodes have a necromenic lifestyle is based on studies that have sought Pristionchus only by sampling live beetles. By surveying for nematode genera in different types of rotting vegetal matter, we found Pristionchus spp. at a similar high frequency as Caenorhabditis, often in large numbers and in feeding stages. Thus, these Pristionchus species may feed in decomposing vegetal matter. In addition, we report that one species of Panagrellus (Nematoda: Panagrolaimidae), Panagrellus redivivoides, is found in rotting fruits but not in rotting stems, with a likely association with Drosophila fruitflies. Based on our sampling and the observed distribution of feeding and dauer stages, we propose a life cycle for Pristionchus nematodes and Panagrellus redivivoides that is similar to that of C. elegans, whereby they feed on the microbial blooms on decomposing vegetal matter and are transported between food patches by coleopterans for Pristionchus spp., fruitflies for Panagrellus redivivoides and isopods and terrestrial molluscs for C. elegans.

Modulation of Gq-Rho Signaling by the ERK MAPK Pathway Controls Locomotion in Caenorhabditis elegans.

The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cß, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of, or in parallel to, the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.

Dopamine negatively modulates the NCA ion channels in C. elegans.

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Genetics: Master Regulator or Master of Disguise?

The pha-1 gene of Caenorhabditis elegans was originally heralded as a master regulator of organ differentiation. A new study suggests instead that pha-1 actually serves no role in development and instead is a component of a selfish genetic element.

The dense-core vesicle maturation protein CCCP-1 binds RAB-2 and membranes through its C-terminal domain.

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here, we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. In addition, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.

The SEK-1 p38 MAP Kinase Pathway Modulates Gq Signaling in Caenorhabditis elegans.

Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling, we performed a forward genetic screen in Caenorhabditis elegans for suppressors of activated Gq. One of the suppressors is an allele of sek-1, which encodes a mitogen-activated protein kinase kinase (MAPKK) in the p38 MAPK pathway. Here, we show that sek-1 mutants have a slow locomotion rate and that sek-1 acts in acetylcholine neurons to modulate both locomotion rate and Gq signaling. Furthermore, we find that sek-1 acts in mature neurons to modulate locomotion. Using genetic and behavioral approaches, we demonstrate that other components of the p38 MAPK pathway also play a positive role in modulating locomotion and Gq signaling. Finally, we find that mutants in the SEK-1 p38 MAPK pathway partially suppress an activated mutant of the sodium leak channel, NCA-1/NALCN, a downstream target of Gq signaling. Our results suggest that the SEK-1 p38 pathway may modulate the output of Gq signaling through NCA-1(unc-77).

The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis elegans.

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here, we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq-Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

Cytoplasmic-Nuclear Incompatibility Between Wild Isolates of Caenorhabditis nouraguensis.

How species arise is a fundamental question in biology. Species can be defined as populations of interbreeding individuals that are reproductively isolated from other such populations. Therefore, understanding how reproductive barriers evolve between populations is essential for understanding the process of speciation. Hybrid incompatibility (for example, hybrid sterility or lethality) is a common and strong reproductive barrier in nature. Here we report a lethal incompatibility between two wild isolates of the nematode Caenorhabditis nouraguensis Hybrid inviability results from the incompatibility between a maternally inherited cytoplasmic factor from each strain and a recessive nuclear locus from the other. We have excluded the possibility that maternally inherited endosymbiotic bacteria cause the incompatibility by treating both strains with tetracycline and show that hybrid death is unaffected. Furthermore, cytoplasmic-nuclear incompatibility commonly occurs between other wild isolates, indicating that this is a significant reproductive barrier within C. nouraguensis We hypothesize that the maternally inherited cytoplasmic factor is the mitochondrial genome and that mitochondrial dysfunction underlies hybrid death. This system has the potential to shed light on the dynamics of divergent mitochondrial-nuclear coevolution and its role in promoting speciation.