A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

Phospholipase C-epsilon regulates epidermal morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3))-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3) production is stimulated is unknown. IP(3) is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3) receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon.     

Small GTPases promote actin coat formation on microsporidian pathogens traversing the apical membrane of Caenorhabditis elegans intestinal cells

Many intracellular pathogens co‐opt actin in host cells, but little is known about these interactions in vivo. We study the in vivo trafficking and exit of the microsporidian Nematocida parisii, which is an intracellular pathogen that infects intestinal cells of the nematode Caenorhabditis elegans. We recently demonstrated that N. parisii uses directional exocytosis to escape out of intestinal cells into the intestinal tract. Here, we show that an intestinal‐specific isoform of C. elegans actin called ACT‐5 forms coats around membrane compartments that contain single exocytosing spores, and that these coats appear to form after fusion with the apical membrane. We performed a genetic screen for host factors required for actin coat formation and identified small GTPases important for this process. Through analysis of animals defective in these factors, we found that actin coats are not required for pathogen exit although they may boost exocytic output. Later during infection, we find that ACT‐5 also forms coats around membrane‐bound vesicles that contain multiple spores. These vesicles are likely formed by clathrin‐dependent compensatory endocytosis to retrieve membrane material that has been trafficked to the apical membrane as part of the exocytosis process. These findings provide insight into microsporidia interaction with host cells, and provide novel in vivo examples of the manner in which intracellular pathogens co‐opt host actin during their life cycle.     

Socket cells mediate spicule morphogenesis in Caenorhabditis elegans males.

Caenorhabditis elegans male spicule morphogenesis requires the coordinated cellular behaviors of several types of cells. We found that the spicule neurons and sheath cells, although important for spicule function, are dispensable for spicule morphology. In contrast, the spicule socket cells are essential for both spicule elongation and formation of spicule cuticle. The socket cells are not only necessary but also sufficient to produce spicule cuticle. This functional aspect of socket cells is genetically separable from their function in mediating spicule elongation: elongated spicules with defective spicule cuticle can be formed. During spicule morphogenesis, the expression of an egl-17::GFP reporter gene is found in the spicule socket cells and its expression appears to be regulated in the socket cells. Mutants defective in TGF-beta signaling display a crumpled spicules phenotype as a result of failure of socket cell movement during spicule morphogenesis. These observations suggest that both the FGF and the TGF-beta signaling pathways might be involved in spicule elongation.     

A novel kinase regulates dietary restriction‐mediated longevity in Caenorhabditis elegans

Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat‐2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3‐like kinase gene, mekk‐3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR‐like state results in upregulation of beta‐oxidation genes through the nuclear hormone receptor NHR‐49, a HNF‐4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta‐oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA‐4/FOXA, NHR‐8 and aryl hydrocarbon receptor AHR‐1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk‐3 knock‐down. Thus, MEKK‐3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk‐3 may signal an imminent nutrient crisis that results in initiation of a DR‐like state, even when food is plentiful.     

Inositol 1,4,5-trisphosphate signaling regulates rhythmic contractile activity of myoepithelial sheath cells in Caenorhabditis elegans

Intercellular communication between germ cells and neighboring somatic cells is essential for reproduction. Caenorhabditis elegans oocytes are surrounded by and coupled via gap junctions to smooth muscle-like myoepithelial sheath cells. Rhythmic sheath cell contraction drives ovulation and is triggered by a factor secreted from oocytes undergoing meiotic maturation. We demonstrate for the first time that signaling through the epidermal growth factor-like ligand LIN-3 and the LET-23 tyrosine kinase receptor induces ovulatory contractions of sheath cells. Reduction-of-function mutations in the inositol 1,4,5-trisphosphate (IP(3)) receptor gene itr-1 and knockdown of itr-1 expression by RNA interference inhibit sheath contractile activity. itr-1 gain-of-function mutations increase the rate and force of basal contractions and induce tonic sheath contraction during ovulation. Sheath contractile activity is disrupted by RNAi of plc-3, one of six phospholipase C-encoding genes in the C. elegans genome. PLC-3 is a PLC-gamma homolog and is expressed in contractile sheath cells of the proximal gonad. Maintenance of sheath contractile activity requires plasma membrane Ca(2+) entry. We conclude that IP(3) generated by LET-23 mediated activation of PLC-gamma induces repetitive intracellular Ca(2+) release that drives rhythmic sheath cell contraction. Calcium entry may function to trigger Ca(2+) release via IP(3) receptors and/or refill intracellular Ca(2+) stores.     

Actin-based forces driving embryonic morphogenesis in Caenorhabditis elegans

Morphogenesis is the process by which multicellular organisms transform themselves from a ball of cells into an organized animal. Certain virtues of Caenorhabditis elegans make it an excellent model system for the study of this process: it is genetically tractable, develops as a transparent embryo with small cell-numbers, and yet still contains all the major tissues typical of animals. Furthermore, certain morphogenetic events are also amenable to study by direct manipulation of the cells involved. Given these advantages, it has been possible to use C. elegans to investigate the different ways in which the actin cytoskeleton drives the cellular rearrangements underlying morphogenesis, through regulated polymerization or actomyosin contraction. Recent insights from this system have determined the involvement in morphogenesis of key proteins, including the actin-regulating WASP and Ena proteins, potential guidance molecules such as the Eph and Robo receptors, and the cell-cell signaling proteins of the Wnt pathway.     

ACT-5 is an essential Caenorhabditis elegans actin required for intestinal microvilli formation

Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.     

Wild-type and mutant actin genes in Caenorhabditis elegans

We have sequenced the four actin genes of Caenorhabditis elegans. These four genes encode typical invertebrate actins and are highly homologous, differing from each other by, at most, three amino acid residues. As a first step toward an understanding of the developmental regulation of this gene set we have also sequenced mutant actin genes. The mutant genes were cloned from two independent revertants of a single dominant actin mutant. For both revertants, reversion was accompanied by an actin gene rearrangement. The accumulation of actin mRNA during development in these two revertants is different from that of wild-type animals. We present here a correlation between actin gene structure and expression in wild-type and mutant animals. The results, suggest that co-ordinate regulation of actin genes is not essential for wild-type muscle function. In addition, it appears that changes in the 3' region of at least one of the actin mRNA may affect its steady-state regulation during development.     

Phospholipase Cepsilon regulates ovulation in Caenorhabditis elegans

Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation.     

Caenorhabditis elegans PlexinA, PLX-1, interacts with transmembrane semaphorins and regulates epidermal morphogenesis

The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos, including axonal guidance. We have generated and characterized putative null mutants of the C. elegans plexinA gene, plx-1. plx-1 mutants exhibited morphological defects: displacement of ray 1 and discontinuous alae. The epidermal precursors for the affected organs were aberrantly arranged in the mutants, and a plx-1::gfp transgene was expressed in these epidermal precursor cells as they underwent dynamic morphological changes. Suppression of C. elegans transmembrane semaphorins, Ce-Sema-1a and Ce-Sema-1b, by RNA interference caused a displacement of ray 1 similar to that of plx-1 mutants, whereas mutants for the Ce-Sema-2a/mab-20 gene, which encodes a secreted-type semaphorin, exhibited phenotypes distinct from those of plx-1 mutants. A heterologous expression system showed that Ce-Sema-1a, but not Ce-Sema-2a, physically bound to PLX-1. Our results indicate that PLX-1 functions as a receptor for transmembrane-type semaphorins, and, though Ce-Sema-2a and PLX-1 both play roles in the regulation of cellular morphology during epidermal morphogenesis, they function rather independently.     

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.     

A novel mode of ecdysozoan growth in Caenorhabditis elegans

Whereas growth in many ecdysozoa is associated with only molting, larval growth in nematodes, specifically Caenorhabditis elegans, is thought to be continuous and exponential. However, this has never been closely investigated. Here we report several detailed studies of growth in wild-type and dwarf C. elegans strains. We find that apparent exponential growth between hatching and adulthood comprises a series of linear phases, one per larval stage, with the linear growth rate increasing at successive molts. Although most structures grow continuously, the buccal cavity does not; instead, it grows saltationally at molts, like arthropod structures. We speculate that these saltational changes in mouth size permit changes in growth rate and that molting exists in nematodes to facilitate rapid growth. We study the cellular basis of this growth in the hypodermis. At each larval stage, lateral seam cells produce daughters that fuse with hyp7, a syncytium covering most of the worm. We find that seam cells and fusing daughter cells obtain larger sizes in successive molts. The total seam cell volume remains constant relative to the size of the worm. However, fusing daughter cells contributes only a very small amount directly to hypodermal growth, suggesting that most hyp7 growth must be intrinsic. Thus, dwarfism mutations studied principally act via adult syncytial growth, with cell size being near normal in both dbl-1 and dpy-2 mutant worms. We speculate that the main function of seam cell proliferation may be to supply the hypodermis with additional genomes for the purpose of growth.     

Sperm status regulates sexual attraction in Caenorhabditis elegans

Mating behavior of animals is regulated by the sensory stimuli provided by the other sex. Sexually receptive females emit mating signals that can be inhibited by male ejaculate. The genetic mechanisms controlling the release of mating signals and encoding behavioral responses remain enigmatic. Here we present evidence of a Caenorhabditis elegans hermaphrodite-derived cue that stimulates male mating-response behavior and is dynamically regulated by her reproductive status. Wild-type males preferentially mated with older hermaphrodites. Increased sex appeal of older hermaphrodites was potent enough to stimulate robust response from mating-deficient pkd-2 and lov-1 polycystin mutant males. This enhanced response of pkd-2 males toward older hermaphrodites was independent of short-chain ascaroside pheromones, but was contingent on the absence of active sperm in the hermaphrodites. The improved pkd-2 male response toward spermless hermaphrodites was blocked by prior insemination or by genetic ablation of the ceh-18-dependent sperm-sensing pathway of the hermaphrodite somatic gonad. Our work suggests an interaction between sperm and the soma that has a negative but reversible effect on a hermaphrodite-derived mating cue that regulates male mating response, a phenomenon to date attributed to gonochoristic species only.     

Goalpha regulates volatile anesthetic action in Caenorhabditis elegans

To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the alpha-subunit of Go, have EC(50)s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goalpha, and presynaptic Goalpha-effectors are candidate VA molecular targets.     

CDC-25.1 regulates germline proliferation in Caenorhabditis elegans

The cell cycles in C. elegans are tightly controlled but appear to use the same regulators found in other organisms. Four homologues of the dual-specificity phosphatase Cdc25 are present in the C. elegans genome. In our study, we have characterized a deletion mutant for one of these orthologues. We show that embryonic defects are absent in cdc-25.1 homozygous mutants, presumably because of maternally contributed CDC-25.1 product. These embryos hatch and develop into sterile adults. The adults do not appear to have any somatic defects. The sterility results from inadequate germline proliferation. Germline precursors divide slowly and produce abnormally sized daughter cells. Only three to four rounds of germ-cell division occur before they die during the L3 and L4 larval stages.     

Tyrosylprotein sulfotransferase regulates collagen secretion in Caenorhabditis elegans

The sulfation of tyrosine residues is an important post-translational modification involved in the regulation of protein function. We examined the activity of worm tyrosylprotein sulfotransferase (TPST-1) on a typical cuticle collagen, ROL-6, in C. elegans. We verified that TPST-1 sulfates three tyrosine residues of ROL-6 in vitro. We found that these tyrosine residues are important for the secretion of ROL-6::GFP. Mutant ROL-6::GFP proteins that contain more than two substitutions of the target tyrosine residues are severely deficient in cuticle localization. Consistently, knock down of tpst-1 blocked the cuticle localization of ROL-6::GFP. Therefore, the sulfation of ROL-6 by TPST-1 is critical for the proper localization of ROL-6. We also confirmed that worm TPST-1 is localized to the trans-Golgi network (TGN). Our results indicate that TPST-1 regulates cuticle organization by promoting the transport of ROL-6 from the TGN to the cuticle.