Genetic analysis of lysosomal trafficking in Caenorhabditis elegans

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.     

Genetic analysis of the heparan modification network in Caenorhabditis elegans

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.     

Genetic analysis of growth cone migrations in Caenorhabditis elegans

Model organisms like Caenorhabditis elegans allow the study of growth cone motility and guidance in vivo. We are using circumferential axon guidance in C. elegans to study both the mechanisms of guidance and the interactions between different guidance systems in vivo. A genetic screen has identified suppressors of the specific axon guidance defects caused by ectopic expression of UNC-5, the repulsive receptor for the UNC-6/netrin guidance cue. These mutations identify eight genes whose products are required for the function of UNC-5 in these cells. In principle, the functions of some of these genes may involve unc-73, which encodes a multidomain, cytoplasmic protein that is an activator of the rac and rho GTPases. Loss of UNC-73 causes errors in axon guidance, and it is hypothesized that UNC-73 acts in multiple signaling pathways used by guidance receptors on the growth cone surface to regulate the underlying cytoskeleton. Here we summarize and discuss these recent developments that are advancing our understanding of growth cone signal transduction in vivo.     

The lin-12 locus of Caenorhabditis elegans

Analysis of the patterns of cell lineage observed during development of the nematode Caenorhabditis elegans, combined with selected cell ablation experiments, has revealed that while many cell fates are autonomously (intrinsically) determined, cell–cell interactions are required for a number of developmental decisions. Earlier genetic analysis of one key gene, lin-12, had shown that this gene controls a number of bi-potential fate decisions involving such cellular interactions. Molecular analysis of this gene is now providing clues to its mode of action in mediating these cell-fate decision.     

Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ-cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.     

Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.     

Genetic and molecular analysis of the dpy-14 region in Caenorhabditis elegans.

Summary Essential genes have been identified in the 1.5 map unit (m.u.)dpy-14-unc-29 region of chromosome I inCaenorhabditis elegans. Previous work defined nine genes with visible mutant phenotypes and nine genes with lethal mutant phenotypes. In this study, we have identified an additional 28 essential genes with 97 lethal mutations. The mutations were mapped using eleven duplication breakpoints, eight deficiencies and three-factor recombination experiments. Genes required for the early stages of development were common, with 24 of the 37 essential genes having mutant phenotypes arresting at an early larval stage. Most mutants of a gene have the same time of arrest; only four of the 20 essential genes with multiple alleles have alleles with different phenotypes. From the analysis of complementing alleles oflet-389, alleles with the same time-of-arrest phenotype were classified as either hypomorphic or amorphic. Mutants oflet-605, let-534 andunc-37 have both uncoordinated and lethal phenotypes, suggesting that these genes are required for the coordination of movement and for viability. The physical and genetic maps in thedpy-14 region were linked by positioning two N2/BO polymorphisms with respect to duplications in the region, and by localizing the right breakpoint of the deficiencyhDf8 on the physical map. Using cross-species hybridization toC. briggsae, ten regions of homology have been identified, eight of which are known to be coding regions, based on Northern analysis and/or the isolation of cDNA clones.     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

Structural and biological characterisation of the gut-associated cyclophilin B isoforms from Caenorhabditis elegans

The free-living nematode Caenorhabditis elegans expresses 18 cyclophilin isoforms, eight of which are conserved single domain forms, comprising two closely related secreted or type B forms (CYP-5 and CYP-6). Recombinant CYP-5 has been purified, crystallised and the X-ray structure solved to a resolution of 1.75A. The detailed molecular architecture most strongly resembles the structure of human cyclophilin B with conserved changes in loop structure and N and C-terminal extensions. Interestingly, the active site pocket is occupied by a molecule of dithiothreitol though this has little effect on the geometry of the active site which is similar to other cyclophilin structures. The peptidyl-prolyl isomerase activity of CYP-5 has been characterised against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 3.6x10(6)M(-1)s(-1) that compares with a value of 6.3x10(6)M(-1)s(-1) for human cyclophilin B. The immunosuppressive drug cyclosporin A binds and inhibits CYP-5 with an IC(50) value of 50nM, which is comparable to the value of 84nM found for human cyclophilin B. CYP-6 has 67% sequence identity with CYP-5 and a molecular model was built based on the CYP-5 crystal structure. The model shows that CYP-5 and CYP-6 are likely to have very similar structures, but with a markedly increased number of negative charges distributed around the surface of CYP-6. The spatial expression patterns of the cyclophilin B isoforms were examined using transgenic animals carrying a LacZ reporter fusion to these genes, and both cyp-5 and cyp-6 are found to be expressed in an overlapping fashion in the nematode gut. The temporal expression pattern of cyp-5 was further determined and revealed a constitutive expression pattern, with highest abundance levels being found in the embryo.     

Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity

Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta- tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.     

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.     

Caenorhabditis elegans: A Genetic Guide to Parasitic Nematode Biology

The advent of parasite genome sequencing projects, as well as an increase in biology-directed gene discovery, promises to reveal genes encoding many of the key molecules required for nematode-host interactions. However, distinguishing parasitism genes from those merely required for nematode viability remains a substantial challenge. Although this will ultimately require a functional test in the host or parasite, the free-living nematode Caenorhabditis elegans can be exploited as a heterologous system to determine function of candidate parasitism genes. Studies of C. elegans also have revealed genetic networks, such as the dauer pathway, that may also be important adaptations for parasitism. As a more directed means of identifying parasitism traits, we developed classical genetics for Heterodera glycines and have used this approach to map genes conferring host resistance-breaking phenotypes. It is likely that the C. elegans and H. glycines genomes will be at least partially syntenic, thus permitting predictive physical mapping of H. glycines genes of interest.     

The atypical calpains: evolutionary analyses and roles in Caenorhabditis elegans cellular degeneration

The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.     

The histones of Caenorhabditis elegans: no evidence of stage-specific isoforms. An overview

The nematode Caenorhabditis elegans expresses one species of H2A and one species of H4 molecules, at least two species of H1 (H1.1, H1.2), two species of H2B (H2B.1, H2B.2) and 2-4 species of H3 (H3.1 and H3.3 and an unassigned Ile/Leu microheterogeneity in H3). The study of their primary structures has been completed now and all of them, with the exception of the Ile/Leu microheterogeneity in H3, have been assigned to protein spots on two-dimensional gels. One spot, previously designated H3.2, probably represents C-terminally cleaved H3.1. The relative abundance of the isohistones was essentially the same when derived from either eggs, gravid adults or postreproductive, senescent worms. The degree of post-translational modification, however, particularly acetylation of H2A, H2B and H3 histone species, was reduced at old age.