The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development

Innexins are the proposed structural components of gap junctions in invertebrates. Antibodies that specifically recognize the Caenorhabditis elegans innexin protein INX-3 were generated and used to examine the patterns of inx-3 gene expression and the subcellular sites of INX-3 localization. INX-3 is first detected in two-cell embryos, concentrated at the intercellular interface, and is expressed ubiquitously throughout the cellular proliferation phase of embryogenesis. During embryonic morphogenesis, INX-3 expression becomes more restricted. Postembryonically, INX-3 is expressed transiently in several cell types, while expression in the posterior pharynx persists throughout development. Through immuno-EM techniques, INX-3 was observed at gap junctions in the adult pharynx, providing supporting evidence that innexins are components of gap junctions. An inx-3 mutant was isolated through a combined genetic and immunocytochemical screen. Homozygous inx-3 mutants exhibit defects during embryonic morphogenesis. At the comma stage of early morphogenesis, variable numbers of cells are lost from the anterior of inx-3(lw68) mutants. A range of terminal defects is seen later in embryogenesis, including localized rupture of the hypodermis, failure of the midbody to elongate properly, abnormal contacts between hypodermal cells, and failure of the pharynx to attach to the anterior of the animal.     

The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans

Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.     

Distinct and redundant functions of mu1 medium chains of the AP-1 clathrin-associated protein complex in the nematode Caenorhabditis elegans

In the nematode Caenorhabditis elegans, there exist two micro1 medium chains of the AP-1 clathrin-associated protein complex. Mutations of unc-101, the gene that encodes one of the micro1 chains, cause pleiotropic effects (). In this report, we identified and analyzed the second mu1 chain gene, apm-1. Unlike the mammalian homologs, the two medium chains are expressed ubiquitously throughout development. RNA interference (RNAi) experiments with apm-1 showed that apm-1 and unc-101 were redundant in embryogenesis and in vulval development. Consistent with this, a hybrid protein containing APM-1, when overexpressed, rescued the phenotype of an unc-101 mutant. However, single disruptions of apm-1 or unc-101 have distinct phenotypes, indicating that the two medium chains may have distinct functions. RNAi of any one of the small or large chains of AP-1 complex (sigma1, beta1, or gamma) showed a phenotype identical to that caused by the simultaneous disruption of unc-101 and apm-1, but not that by single disruption of either gene. This suggests that the two medium chains may share large and small chains in the AP-1 complexes. Thus, apm-1 and unc-101 encode two highly related micro1 chains that share redundant and distinct functions within AP-1 clathrin-associated protein complexes of the same tissue.     

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.     

EXT gene family member rib-2 is essential for embryonic development and heparan sulfate biosynthesis in Caenorhabditis elegans

EXT gene family members including EXT1, EXT2, and EXTL2 are glycosyltransferases required for heparan sulfate biosynthesis. To examine the biological functions of rib-2, a member of the Caenorhabditis elegans EXT gene family, we generated a mutant worm lacking the rib-2 gene using the UV-TMP method followed by sib-selection. Inactivation of rib-2 alleles induced developmental abnormalities in F2 and F3 homozygous worms, while F1 heterozygotes showed a normal morphology. The F2 homozygous progeny generated from the F1 heterozygous hermaphrodites somehow developed to adult stage but exhibited abnormal characteristics such as developmental delay and egg-laying defects. The F3 homozygous progeny from the F2 homozygous hermaphrodites showed early developmental defects and most of the F3 worms stopped developing during the gastrulation stage. Whole-mount staining analysis for heparan sulfate using Toluidine blue (pH 2.5) revealed a defect of heparan sulfate biosynthesis in the F2 homozygotes. The analysis using fluorometric post-column high-performance liquid chromatography also uncovered reduced production of heparan sulfate in the rib-2 mutant. These results indicate that rib-2 is essential for embryonic development and heparan sulfate biosynthesis in C. elegans.     

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.     

Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans.

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.     

The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78-null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1-null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.     

Essential roles of 3'-phosphoadenosine 5'-phosphosulfate synthase in embryonic and larval development of the nematode Caenorhabditis elegans

Sulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells.     

The Caenorhabditis elegans septin complex is nonpolar

Septins are conserved GTPases that form heteromultimeric complexes and assemble into filaments that play a critical role in cell division and polarity. Results from budding and fission yeast indicate that septin complexes form around a tetrameric core. However, the molecular structure of the core and its influence on the polarity of septin complexes and filaments is poorly defined. The septin complex of the nematode Caenorhabditis elegans is formed entirely by the core septins UNC-59 and UNC-61. We show that UNC-59 and UNC-61 form a dimer of coiled-coil-mediated heterodimers. By electron microscopy, this heterotetramer appears as a linear arrangement of four densities representing the four septin subunits. Fusion of GFP to the N termini of UNC-59 and UNC-61 and subsequent electron microscopic visualization suggests that the sequence of septin subunits is UNC-59/UNC-61/UNC-61/UNC-59. Visualization of GFP extensions fused to the extremity of the C-terminal coiled coils indicates that these extend laterally from the heterotetrameric core. Together, our study establishes that the septin core complex is symmetric, and suggests that septins form nonpolar filaments.     

Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development

Caenorhabditis elegans germ cell proliferation and development were severely damaged in second generation dna-2 homozygotes. Even in the first generation, a much higher incidence of aberrant chromosomes in oocytes and resultantly higher embryonic lethality were found vs. wild type, when DNA breaks were induced by gamma-rays or camptothecin. The deficiency of dna-2 in combination with RNA interference on mre-11 gene expression synergistically aggravated germ-line development, especially oocyte formation. These results suggest that C. elegans Dna-2 is involved in a DNA repair pathway paralleling homologous recombination or non-homologous end joining with mre-11 participation.     

CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans

The Caenorhabditis elegans distal tip cells (DTCs) are an in vivo model for the study of developmentally regulated cell migration. In this study, we characterize a novel role for CCDC-55, a conserved coiled-coil domain containing protein, in DTC migration and larval development in C. elegans. Although animals homozygous for a probable null allele, ccdc-55(ok2851), display an early larval arrest, RNAi depletion experiments allow the analysis of later phenotypes and suggest that CCDC-55 is needed within the DTC for migration to cease at the end of larval morphogenesis. The ccdc-55 gene is found in an operon with rnf-121 and rnf-5, E3 ubiquitin ligases that target cell migration genes such as the β-integrin PAT-3. Genetic interaction studies using RNAi depletion and the deletion alleles rnf-121(ok848) and rnf-5(tm794) indicate that CCDC-55 and the RNF genes act at least partially in parallel to promote termination of cell migration in the adult DTC.     

TBC-2 is required for embryonic yolk protein storage and larval survival during L1 diapause in Caenorhabditis elegans

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause.     

The L-isoaspartyl-O-methyltransferase in Caenorhabditis elegans larval longevity and autophagy

The protein L-isoaspartyl-O-methyltransferase, coded by the pcm-1 gene in Caenorhabditis elegans, participates in the repair of age-damaged proteins. We tested the ability of pcm-1-deficient nematodes to survive starvation stress as developmentally-arrested L1 larvae. We found that pcm-1 mutant L1 larvae do not survive as well as wild-type L1 larvae when incubated in M9 medium without nutrients. We then tested whether the starved L1 larvae could continue development when allowed access to food in a recovery assay. A loss of recovery ability with age was observed for all larvae, with little or no difference between the pcm-1 mutant and wild-type N2 larvae. Interestingly, when L1 larvae were starved in cholesterol-containing S medium or M9 medium supplemented with cholesterol, the survival rates of both mutant and wild-type animals nearly doubles, with pcm-1 larvae again faring more poorly than N2 larvae. Furthermore, L1 larvae cultured in these cholesterol-containing media show an increase in Sudan Black staining over animals cultured in M9 medium. The longevity defects of pcm-1 mutants previously seen in dauer larvae and here in L1 larvae suggest a defect in the ability of pcm-1 mutants to recycle and reuse old cellular components in pathways such as autophagy. Using an autophagosomal marker, we found evidence suggesting that the pcm-1 mutation may inhibit autophagy during dauer formation, suggesting that the absence of protein repair may also interfere with protein degradation pathways.     

Caenorhabditis elegans lin-45 raf is essential for larval viability, fertility and the induction of vulval cell fates

The protein kinase Raf is an important signaling protein. Raf activation is initiated by an interaction with GTP-bound Ras, and Raf functions in signal transmission by phosphorylating and activating a mitogen-activated protein (MAP) kinase kinase named MEK. We identified 13 mutations in the Caenorhabditis elegans lin-45 raf gene by screening for hermaphrodites with abnormal vulval formation or germline function. Weak, intermediate, and strong loss-of-function or null mutations were isolated. The phenotype caused by the most severe mutations demonstrates that lin-45 is essential for larval viability, fertility, and the induction of vulval cell fates. The lin-45(null) phenotype is similar to the mek-2(null) and mpk-1(null) phenotypes, indicating that LIN-45, MEK-2, and MPK-1 ERK MAP kinase function in a predominantly linear signaling pathway. The lin-45 alleles include three missense mutations that affect the Ras-binding domain, three missense mutations that affect the protein kinase domain, two missense mutations that affect the C-terminal 14-3-3 binding domain, three nonsense mutations, and one small deletion. The analysis of the missense mutations indicates that Ras binding, 14-3-3-binding, and protein kinase activity are necessary for full Raf function and suggests that a 14-3-3 protein positively regulates Raf-mediated signaling during C. elegans development.     

The Caenorhabditis elegans ortholog of human PHF5a shows a muscle-specific expression domain and is essential for C. elegans morphogenetic development

We have recently described a novel human and murine multigene-family that is highly conserved during evolution and shows a PHD-finger-like domain present in the deduced protein sequences. Here, we describe the cloning and characterization of the Caenorhabditis elegans ortholog of human PHF5a. Transgenic phf-5::yfp-reporter techniques in C. elegans identified temporal C. elegans phf-5 expression being restricted to late C. elegans development. The phf-5::yfp expression starts within the morphogenetic phase of embryonic development and lasts to the stage of adult worms. Spatial phf-5 expression is muscle-specific with an expression in the developing pharynx, in body wall muscular structures, and in the anal muscles. By phf-5 RNAi we further demonstrated that PHF-5 is essential in the morphogenetic phase of C. elegans embryonic development as well as in young larvae. In contrast, phf-5 RNAi does not show an evident phenotype to adult worms. Taken together, this is the first report providing evidence for a tissue and stage-specific expression of a PHF5a ortholog, named phf-5, in C. elegans while our data further suggest an essential role of the encoded PHF-5 protein in morphogenetic development and muscle function.     

Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.