Deciphering endocytosis in Caenorhabditis elegans

We discuss in this review recent studies using the worm Caenorhabditis elegans to decipher endocytic trafficking in a multicellular organism. Recent advances, including in vivo assay systems, new genetic screens, comparative functional analysis of conserved proteins, and RNA-mediated interference (RNAi) in C. elegans, are being used to study the functions of known membrane trafficking factors and to identify new ones. The ability to monitor endocytosis in vivo in worms allows one to test current endocytosis models and to demonstrate the physiological significance of factors identified by genetic and biochemical methods. The available human genome sequence facilitates comparative studies where human homologs of new factors identified in C. elegans can be quickly assayed for similar function using traditional cell biological methods in mammalian cell systems. New studies in C. elegans have used a combination of these techniques to reveal novel metazoan-specific trafficking factors required for endocytosis. Many more metazoan-specific trafficking factors and insights into the mechanisms of endocytosis are likely to be uncovered by analysis in C. elegans .     

Caenorhabditis elegans mutant allele identification by whole-genome sequencing

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.     

Genome-wide prediction of genetic interactions in a metazoan

Genetic interactions provide information about genes and processes with overlapping functions in biological systems. For Saccharomyces cerevisiae, computational integration of multiple types of functional genomic data is used to generate genome-wide predictions of genetic interactions. However, this methodology cannot be applied to the vastly more complex genome of metazoans, and only recently has the first metazoan genome-wide prediction of genetic interactions been reported. The prediction for Caenorhabditis elegans was generated by computationally integrating functional genomic data from S. cerevisiae, C. elegans and Drosophila melanogaster. This achievement is an important step toward system-level understanding of biological systems and human diseases.     

Informatics issues in large-scale sequence analysis: elucidating the protein kinases of C. elegans

With the availability of the nearly complete genomic sequence of C. elegans, the first multicellular organism to be sequenced, molecular biology has definitely entered the postgenomic era. Annotation of the genomic sequence, which refers to identifying the genes and other biologically relevant sections of the genome, is an important and nontrivial next step. A first-pass annotation will be necessarily incomplete but will drive further biological experiments, which in turn will help to annotate the genome better. Given the scale of the genome sequence analysis, it is clear that the annotation should be automated as much as possible without sacrificing the quality of analysis. In this work, we outline our approach to identifying the protein kinases of C. elegans from the genomic sequence. We describe new tools we have developed for analysis, management and visualization of genomic data. By developing modular and scalable solutions, this study has provided a framework for future analysis of the Drosophila and human genomes.     

Sequence annotation of nuclear receptor ligand-binding domains by automated homology modeling

The quality of three-dimensional homology models derived from protein sequences provides an independent measure of the suitability of a protein sequence for a certain fold. We have used automated homology modeling and model assessment tools to identify putative nuclear hormone receptor ligand-binding domains in the genome of Caenorhabditis elegans. Our results indicate that the availability of multiple crystal structures is crucial to obtaining useful models in this receptor family. The majority of annotated mammalian nuclear hormone receptors could be assigned to a ligand-binding domain fold by using the best model derived from any of four template structures. This strategy also assigned the ligand-binding domain fold to a number of C.elegans. sequences without prior annotation. Interestingly, the retinoic acid receptor crystal structure contributed most to the number of sequences that could be assigned to a ligand-binding domain fold. Several causes for this can be suggested, including the high quality of this protein structure in terms of our assessment tools, similarity between the biological function or ligand of this receptor and the modeled genes and gene duplication in C.elegans.     

The relative roles of three DNA repair pathways in preventing Caenorhabditis elegans mutation accumulation

Mutation is a central biological process whose rates and spectra are influenced by a variety of complex and interacting forces. Although DNA repair pathways are generally known to play key roles in maintaining genetic stability, much remains to be understood about the relative roles of different pathways in preventing the accumulation of mutations and the extent of heterogeneity in pathway-specific repair efficiencies across different genomic regions. In this study we examine mutation processes in base excision repair-deficient (nth-1) and nucleotide excision repair-deficient (xpa-1) Caenorhabditis elegans mutation-accumulation (MA) lines across 24 regions of the genome and compare our observations to previous data from mismatch repair-deficient (msh-2 and msh-6) and wild-type (N2) MA lines. Drastic variation in both average and locus-specific mutation rates, ranging two orders of magnitude for the latter, was detected among the four sets of repair-deficient MA lines. Our work provides critical insights into the relative roles of three DNA repair pathways in preventing C. elegans mutation accumulation and provides evidence for the presence of pathway-specific DNA repair territories in the C. elegans genome.     

CRISPR-Cas9基因编辑技术在秀丽线虫中的应用

基于细菌基因组规律成蔟的间隔短回文重复(Clustered regularly interspaced short palindromic repeats)发展而来的新型基因编辑方法(CRISPR-Cas9)对生物医学研究是一场划时代的革命。它几乎可用于大多数生物体的基因编辑。秀丽线虫是一种非常经典的遗传学模式生物,CRISPR-Cas9基因编辑技术进一步加速了对其基因功能及各种生物学问题的研究。文中主要总结CRISPR-Cas9基因编辑系统在遗传学模式生物秀丽线虫中的发展和应用。     

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.     

Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-box proteins

BACKGROUND: The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. RESULTS: Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities. CONCLUSIONS: The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans.     

Evolutionary history of Caenorhabditis elegans inferred from microsatellites: evidence for spatial and temporal genetic differentiation and the occurrence of outbreeding

Although diverse biological disciplines employ the nematode Caenorhabditis elegans as a highly efficient laboratory model system, little is known about its natural history. We investigated its evolutionary past using 10 polymorphic trinucleotide and tetranucleotide microsatellites, derived from across the whole genome. These microsatellites were analyzed from the 35 previously available natural isolates from different parts of the world and also 23 new strains isolated from northwest Germany. Our results highlight that C. elegans lineages differentiate genetically with respect to geographic distance and, to a lesser extent, differences in the time of strain isolation. The latter indicates some turnover of strain genotypes at specific locations. Our data also demonstrate the coexistence of highly diverse genotypes in the population from northwest Germany, which is best explained by recent migration events. Furthermore, selfing is confirmed as the primary mode of reproduction for this hermaphroditic nematode in nature. Importantly, we also find evidence for the occurrence of occasional outbreeding. Taken together, these results support the previous notion that C. elegans is a colonizer, whereby selfing may permit rapid dispersal within new habitats even in the absence of potential mates, whereas occasional outcrossing may serve to compensate for the disadvantages of inbreeding. Such information about the natural history of C. elegans should be of great value for an in-depth understanding of the complexity of this organism, including its multifaceted developmental, neurological, or molecular genetic pathways.     

MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline

Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3'UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.     

Evidence for active maintenance of inverted repeat structures identified by a comparative genomic approach

Inverted repeats have been found to occur in both prokaryotic and eukaryotic genomes. Usually they are short and some have important functions in various biological processes. However, long inverted repeats are rare and can cause genome instability. Analyses of C. elegans genome identified long, nearly-perfect inverted repeat sequences involving both divergently and convergently oriented homologous gene pairs and complete intergenic sequences. Comparisons with the orthologous regions from the genomes of C. briggsae and C. remanei show that the inverted repeat structures are often far more conserved than the sequences. This observation implies that there is an active mechanism for maintaining the inverted repeat nature of the sequences.     

WormBase: a cross-species database for comparative genomics

WormBase (http://www.wormbase.org/) is a web-accessible central data repository for information about Caenorhabditis elegans and related nematodes. The past two years have seen a significant expansion in the biological scope of WormBase, including the integration of large-scale, genome-wide data sets, the inclusion of genome sequence and gene predictions from related species and active literature curation. This expansion of data has also driven the development and refinement of user interfaces and operability, including a new Genome Browser, new searches and facilities for data access and the inclusion of extensive documentation. These advances have expanded WormBase beyond the obvious target audience of C. elegans researchers, to include researchers wishing to explore problems in functional and comparative genomics within the context of a powerful genetic system.     

The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cell proliferation

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.     

A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.     

Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity.

Formation of cross-linking between proteins via a gamma-glutamyl-epsilon-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.