VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes

There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.     

Mutant sensory cilia in the nematode Caenorhabditis elegans

Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.     

CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells

BACKGROUND: Polarised trafficking of proteins is critical for normal expression of the epithelial phenotype, but its genetic control is not understood. The regulatory gene lin-26 is essential for normal epithelial differentiation in the nematode Caenorhabditis elegans. To identify potential effectors of lin-26, we characterised mutations that result in lin-26-like phenotypes. Here, we report the phenotypic and molecular analysis of one such mutant line, che-14. RESULTS: Mutations in che-14 resulted in several partially penetrant phenotypes affecting the function of most epithelial or epithelial-like cells of the ectoderm, including the hypodermis, excretory canal, vulva, rectum and several support cells. The defects were generally linked to the accumulation of vesicles or amorphous material near the apical surface, suggesting that secretion was defective. The CHE-14 protein showed similarity to proteins containing sterol-sensing domains, including Dispatched, Patched and NPC1. A fusion protein between full-length CHE-14 and the green fluorescent protein became localised to the apical surface of epithelial cells that require che-14 function. Deletions that removed the predicted transmembrane domains or extracellular loops of CHE-14 abolished apical localisation and function of the protein. CONCLUSIONS: We propose that CHE-14 is involved in a novel secretory pathway dedicated to the exocytosis of lipid-modified proteins at the apical surface of certain epithelial cells. Our data raise the possibility that the primordial function of proteins containing a sterol-sensing domain is to control vesicle trafficking: CHE-14 and Dispatched in exocytosis, Patched and NPC1 in endocytosis.     

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 "a" subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals.     

Dye-filling of the amphid sheath glia: implications for the functional relationship between sensory neurons and glia in Caenorhabditis elegans

The nervous system is composed of cells including neurons and glia. It has been believed that the former cells play central roles in various neural functions while the latter ones have only supportive functions for neurons. However, recent findings suggest that glial cells actively participate in neural activities, and the cooperation between neurons and glia is important for nervous system functions. In Caenorhabditis elegans, amphid sensory organs in the head also consist of sensory neurons and glia-like support cells (amphid socket and amphid sheath cells). Ciliary endings of some sensory neurons exposed to the environment detect various chemicals, molecules and signals, and the cilia of some neurons can also take up fluorescent dyes such as DiI. Here, we show that the amphid sheath glia are also stained with DiI and that its uptake by the amphid sheath cells correlates with DiI-filling of sensory neurons, suggesting that the amphid sheath glia might interact with sensory neurons. Furthermore, the localization of the amphid sheath cell reporter F52E1.2SP::YFP is abnormal in che-2 mutants, which have defective cilia. These findings imply that sensory neurons might affect amphid sheath glia functions in the amphid sensory organ of C. elegans.     

C. elegans daf-6 encodes a patched-related protein required for lumen formation

Sensory organs are often composed of neuronal sensory endings accommodated in a lumen formed by ensheathing epithelia or glia. Here we show that lumen formation in the C. elegans amphid sensory organ requires the gene daf-6. daf-6 encodes a Patched-related protein that localizes to the luminal surfaces of the amphid channel and other C. elegans tubes. While daf-6 mutants display only amphid lumen defects, animals defective for both daf-6 and the Dispatched gene che-14 exhibit defects in all tubular structures that express daf-6. Furthermore, DAF-6 protein is mislocalized, and lumen morphogenesis is abnormal, in mutants with defective sensory neuron endings. We propose that amphid lumen morphogenesis is coordinated by neuron-derived cues and a DAF-6/CHE-14 system that regulates vesicle dynamics during tubulogenesis.     

Molting-specific downregulation of C. elegans body-wall muscle attachment sites: The role of RNF-5 E3 ligase

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/β-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process.     

Cuticle Formation in Hemicycliophora arenaria,Aphelenchus avenae and HirschmannIella gracilis

The onset of molting in all stages of Hemicycliophora arenaria was preceded by the appearance of numerous, discrete globular structures which were termed "molting bodies" because they were present in the hypodermis only during the production of the new cuticle. In all parasitic stages the molt commenced with the separation of the cuticle from the hypodermis from which the new sheath and cuticle were differentiated. Following completion of the new sheath and cuticle most of the old outer covering was apparently absorbed before ecdysis. Electronmicrographs of body wall cross sections in molting L4 male specimens revealed the final molt to be a double molt in which an additional sixth cuticle was produced. Since both a new sheath and cuticle were produced during the molt of each stage, the sheath must be considered as an integral part of the cuticle and not as a residual cuticle or the result of an incomplete additional molt. Molting in Aphelenchus avenae and Hirschmanniella gracilis was less complex and "molting bodies" were not observed. After cuticle separation the hypodermis gave rise to a new trilaminate zone, the future cortex, and (later) the matrix and striated basal layers.     

Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery

Individual cells of the Caenorhabditis elegans secretory-excretory system were ablated by laser microbeam in various larval stages. Effects on growth, molting, osmoregulation, fertility, longevity, and dauer larva formation were tested. Single-cell ablations did not prevent subsequent molting, but ablation of the pore cell or the duct cell resulted in the absence of the normal cuticular lining of the excretory duct following a molt. When the pore cell, duct cell, or excretory cell was ablated, the animals filled with fluid within 12-24 hr and died within a few days, producing very few progeny. Ablation of the excretory gland cell, on the other hand, had no obvious developmental or behavioral effects. Excretory activity was monitored in dauer larvae by observing pulsation of the excretory duct in conditions of differing osmolarity. The rate of pulsation was quite variable over time in conditions of low osmotic strength, but average five- to six-fold higher than that observed in buffered saline. These observations, combined with the effects of laser ablation, lead to the conclusion that one function of the excretory system is osmoregulation.     

Some, but not all, retromer components promote morphogenesis of C. elegans sensory compartments

The endings of sensory receptor cells often lie within specialized compartments formed by glial cells. The main sensory organ of Caenorhabditis elegans, the amphid, provides a powerful setting for studying glial compartment morphogenesis. Our previous studies showed that amphid compartment size is controlled by opposing activities of the Nemo-like kinase LIT-1, which promotes compartment expansion, and the Patched-related protein DAF-6, which restricts compartment growth. From a genetic screen for mutations able to suppress the bloated sensory compartments of daf-6 mutants, we identified an allele of the sorting nexin gene snx-1. SNX-1 protein is a component of the retromer, a protein complex that facilitates recycling of transmembrane proteins from the endosome to the Golgi network. We find that snx-1 functions cell autonomously within glia to promote sensory compartment growth, and that SNX-1 protein is enriched near the surface of the sensory compartment. snx-1 interacts genetically with lit-1 and another regulator of compartment size, the Dispatched-related gene che-14. Mutations in snx-3 and vps-29, also retromer genes, can suppress daf-6 defects. Surprisingly, however, remaining retromer components seem not to be involved. Our results suggest that a novel assembly of retromer components is important for determining sensory compartment dimensions.     

A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of C. elegans sensory cilia.

To elucidate the mechanism of sensory cilium formation, we analyzed mutants in the Caenorhabditis elegans che-2 gene. These mutants have extremely short cilia with an abnormal posterior projection, and show defects in behaviors that are mediated by ciliated sensory neurons. The che-2 gene encodes a new member of the WD40 protein family, suggesting that it acts in protein-protein interaction. Analysis of mutation sites showed that both the amino-terminal WD40 repeats and the carboxyl-terminal non-WD40 domain are necessary for the CHE-2 function. CHE-2-tagged green fluorescent protein is localized at the cilia of almost all the ciliated sensory neurons. Expression of che-2 in a subset of sensory neurons of a che-2 mutant by using a heterologous promoter resulted in restoration of the functions and cilium morphology of only the che-2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique can be used in the future for determining the function of each type of che-2-expressing sensory neuron. Using green fluorescent protein, we found that the extension of cilia in wild-type animals took place at the late embryonic stage, whereas the cilia of che-2 mutant animals remained always short during development. Hence, the abnormal posterior projection is due to the inability of cilia to extend, rather than degeneration of cilia once correctly formed. Expression of che-2 in a che-2 mutant under a heat shock promoter showed that the extension of cilia, surprisingly, can occur even at the adult stage, and that such cilia can function apparently normally in behavior.     

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.     

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.     

The Ultrastructure of the Presumed Chemoreceptor Aesthetasc "Y" of a Cypridid Ostracode

The aesthetasc "Y" of cypridid ostracodes is divided into three parts: distal, intermediate, and proximal. Distally, it is equipped with five pores containing electron-dense plugs. The distal part shows a peculiar striated pattern formed by two types of cuticle. The intermediate and proximal parts are smooth with no pores or other substructures. 14 cilia run in the large receptor-cavity of the distal part. They originate from 14 bipolar sensory cell bodies within the antenna. From the intermediate part, the cilia are grouped in five cuticular sheaths, surrounded by five sheath cells arranged mesaxonally. At the level of the ciliary junctions, the sheath cells form cavities in which the cilia join the dendrites. Before molting, a new aesthetasc is formed in the antenna by hypodermal cells. The cuticle is secreted in a fold around the sheath cells, which in a way act as moulds. The cilia of the old aesthetasc run through the five pores of the new aesthetasc. The main function of the pores is apparently to enable the animal to obtain information from the surroundings also during the molting period. On morphological grounds, the aesthetasc "Y" is believed to be a chemoreceptor.     

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Distinct rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. We showed that ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Our biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes.     

Fine structure and molting of aesthetasc sense organs on the antennules of the isopod,Asellus aquaticus (Crustacea)

In Asellus aquaticus certain distal antennular segments bear single sensilla referred to as aesthetascs. These show a proximal stem and a distal bulbous region. Depending on its position, each aesthetasc is innervated by either 50-60 or 70-80 bipolar sensory cells, the perikarya of which are situated within the pedunculus. Within the antennular segment the dendrites develop unbranched cilia (9 X 2 + 0 structure). The sensory cells are unusual in that mono- as well as biciliary dendrites are present within a single aesthetasc, the ratio of both types being correlated with the number of sensory cells. Cilia and receptor lymph cavity are enveloped by a set of 3-4 inner and 13-14 outer sheath cells, which terminate at the base of the sensillum, so that the delicate and poreless cuticle of the bulbous region encloses only outer segments within the receptor lymph fluid. A new molting type in arthropods is described in which the outer sheath cells alone build the new cuticle, whereas the inner sheath cells most probably have a protective function. A definition of aesthetascs is proposed based on fine-structural criteria. Functionally the sensilla are considered to be chemoreceptors. This assumption is confirmed by experiments with diluted vital dye as well as lanthanum showing that dissolved substances penetrate the poreless cuticle instantaneously.     

Mutants of Caenorhabditis elegans that form dauer-like larvae

The development, ultrastructure, and genetics of two mutants that form dauer-like larvae have been characterized. Dauer larva morphogenesis is initiated regardless of environmental stimuli, and it is incomplete or abnormal. The resistance to detergent characteristic of normal dauer larvae is not fully achieved, and the mutants are unable to exit from the dauer-like state of developmental arrest. Mutant life span is not extended beyond the three weeks characteristic of the nondauer life cycle, whereas normal dauer larvae can live for several months. Growth of daf-15(m81)IV, the less dauer-like of the two, is nearly arrested at the second (dauer-specific) molt, but feeding is not completely suppressed. Head shape, cuticle, and intestinal ultrastructure are nondauer, whereas sensory structures (amphid and deirid) and excretory gland morphology are intermediate between that of dauer and nondauer stages. The daf-9(e1406)X mutant is dauer-like in head shape, cuticle, and deirid ultrastructure, intermediate in amphid and inner labial neuron morphology, and nondauer or abnormal in the intestine. Also, the daf-9 mutant exhibits abnormalities in the pharyngeal arcade cell processes and pharyngeal g1 gland. Double mutants carrying both daf-9 and daf-15 are more resistant to detergent than either single mutant. Like the single mutants, they cannot complete morphogenesis, and they are unable to exit from the dauer-like stage. Both daf-9 and daf-15 mutations are epistatic to previously described dauer-defective mutations, indicating that these two genes act late in the pathway leading to the dauer larva. The genetic tests and the mutant ultrastructure suggest that the two genes may affect parallel pathways of morphogenesis.