Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.     

Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions

The amenability of Caenorhabditis elegans against pathogen provides a valuable tool for studying host–pathogen interactions. Physiological experiments revealed that the P. aeruginosa was able to kill C. elegans efficiently. The effects of P. aeruginosa PA14, PAO1 and their isolated lipopolysaccharide (LPS) on the host system were analyzed. The LPS at higher concentrations (≥2 mg/ml) was toxic to the host animals. Kinetic studies using qPCR revealed the regulation of host-specific candidate antimicrobial genes during pathogen-mediated infections. In addition, the pathogen-specific virulent gene, exoT expression, was anlyzed and found to be varied during the interactions with the host system. Ability of the pathogens to modify their internal machinery in the presence of the host was analyzed by XRD, FTIR and PCA. LPS isolated from pathogens upon exposure to C. elegans showed modifications at their functional regions. LPS from PAO1 showed difference in d-spacing angle (Å) and °2Th position. FTIR spectra revealed alterations in polysaccharide (1,200–900 cm⁻¹) and fatty acid (3,000–2,800 cm⁻¹) regions of LPS from P. aeruginosa PAO1 exposed to the host system. These data provide additional insights on how the pathogens subvert its own and host machinery during interactions.     

Identification and characterization of Crumbs polarity complex proteins in Caenorhabditis elegans

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.     

Cell interactions involved in development of the bilaterally symmetrical intestinal valve cells during embryogenesis in Caenorhabditis elegans.

We describe two different cell interactions that appear to be required for the proper development of a pair of bilaterally symmetrical cells in Caenorhabditis elegans called the intestinal valve cells. Previous experiments have shown that at the beginning of the 4-cell stage of embryogenesis, two sister blastomeres called ABa and ABp are equivalent in development potential. We show that cell interactions between ABp and a neighboring 4-cell-stage blastomere called P2 distinguish the fates of ABa and ABp by inducing descendants of ABp to produce the intestinal valve cells, a cell type not made by ABa. A second cell interaction appears to occur later in embryogenesis when two bilaterally symmetrical descendants of ABp, which both have the potential to produce valve cells, contact each other; production of the valve cells subsequently becomes limited to only one of the two descendants. This second interaction does not occur properly if the two symmetrical descendants of ABp are prevented from contacting each other. Thus the development of the intestinal valve cells appears to require both an early cell interaction that establishes a bilaterally symmetrical pattern of cell fate and a later interaction that breaks the symmetrical cell fate pattern by restricting to only one of two cells the ability to produce a pair of valve cells.     

Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.     

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.     

Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.     

Requirement of V-ATPase for ovulation and embryogenesis in Caenorhabditis elegans

Immunofluorescence analysis indicated that VHA-11, the C subunit of Caenorhabditis elegans V-ATPase, was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage. Vital staining with acridine orange showed that the intestinal cells had acidic compartments generated by V-ATPase, consistent with the intracellular localization of VHA-11. RNA interference could efficiently silence vha-11 gene expression: introduction of vha-11 double strand RNA led to embryonic lethality. Worms injected with the vha-11 double strand RNA produced embryos that became lethal. The development of embryos was arrested at various stages. However, their numbers gradually decreased, and the worms eventually became sterile due to the failure of ovulation. Similar results were obtained for RNA interference of the V-ATPase proteolipid genes. These results suggest that V-ATPases, and thus inside-acidic organelles, are required for ovulation and embryogenesis.     

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

Analysis of point mutants in the Caenorhabditis elegans vesicular acetylcholine transporter reveals domains involved in substrate translocation

Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans (CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (K(m)), with values ranging from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The relative order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo.     

Neuronal mechanisms of Caenorhabditis elegans and pathogenic bacteria interactions

Individuals interact with environment through different neuronal functions, such as olfaction and mechanosensation; experience shapes these physiological functions. It is not well understood how an individual senses and processes multiple cues of natural stimuli in the environment and how experience modulates these physiological mechanisms. Recent molecular genetics and behavioral studies on the interactions of the genetic model organism Caenorhabditis elegans with pathogenic bacteria have provided insights on the molecular and cellular mechanisms underlying these regulatory processes.     

Autophagy and insulin/TOR signaling in Caenorhabditis elegans pcm-1 protein repair mutants

Biological responses due to nutrient deprivation in the nematode Caenorhabditis elegans, including L1 diapause and autophagy during dauer formation, can be mediated through the linked DAF-2/insulin/IGF receptor and target-of-rapamycin (TOR) kinase pathways. Here we discuss how altered insulin/TOR signaling may underlie the previously reported phenotypes of worms with a null mutation in the pcm-1 gene that results in reduced autophagy during dauer formation and decreased L1 arrest survival. PCM-1 encodes a protein repair methyltransferase and mutants of the encoding pcm-1 gene are incapable of converting spontaneously damaged l-isoaspartyl residues in cellular proteins to normal forms by this pathway. We speculate that PCM-1 may function either directly or indirectly as an inhibitor of insulin/TOR signaling, perhaps in a role to balance autophagy with alternative protein degradation pathways that may be more specific for recognizing age-damaged proteins.     

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.