Guidelines for monitoring autophagy in Caenorhabditis elegans

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.     

Cytoplasmic expression of mouse prion protein causes severe toxicity in Caenorhabditis elegans

To test if Caenorhabditis elegans could be established as a model organism for prion study, we created transgenic C. elegans expressing the cytosolic form of the mouse prion protein, MoPrP(23-231), which lacks the N-terminal signal sequence and the C-terminal glycosylphosphatidylinisotol (GPI) anchor site. We report here that transgenic worms expressing MoPrP(23-231)-CFP exhibited a wide range of distinct phenotypes: from normal growth and development, reduced mobility and development delay, complete paralysis and development arrest, to embryonic lethality. Similar levels of MoPrP(23-231)-CFP were produced in animals exhibiting these distinct phenotypes, suggesting that MoPrP(23-231)-CFP might have misfolded into distinct toxic species. In combining with the observation that mutations in PrP that affect prion pathogenesis also affect the toxic phenotypes in C. elegans, we conclude that the prion protein-folding mechanism is similar in mammals and C. elegans. Thus, C. elegans can be a useful model organism for prion research.     

A Transparent Window into Biology: A Primer on Caenorhabditis elegans

A little over 50 years ago, Sydney Brenner had the foresight to develop the nematode (round worm) Caenorhabditis elegans as a genetic model for understanding questions of developmental biology and neurobiology. Over time, research on C. elegans has expanded to explore a wealth of diverse areas in modern biology including studies of the basic functions and interactions of eukaryotic cells, host–parasite interactions, and evolution. C. elegans has also become an important organism in which to study processes that go awry in human diseases. This primer introduces the organism and the many features that make it an outstanding experimental system, including its small size, rapid life cycle, transparency, and well-annotated genome. We survey the basic anatomical features, common technical approaches, and important discoveries in C. elegans research. Key to studying C. elegans has been the ability to address biological problems genetically, using both forward and reverse genetics, both at the level of the entire organism and at the level of the single, identified cell. These possibilities make C. elegans useful not only in research laboratories, but also in the classroom where it can be used to excite students who actually can see what is happening inside live cells and tissues.     

C. elegans fat storage and metabolic regulation

C. elegans has long been used as an experimentally tractable organism for discovery of fundamental mechanisms that underlie metazoan cellular function, development, neurobiology, and behavior. C. elegans has more recently been exploited to study the interplay of environment and genetics on lipid storage pathways. As an experimental platform, C. elegans is amenable to an extensive array of forward and reverse genetic, a variety of “omics” and anatomical approaches that together allow dissection of complex physiological pathways. This is particularly relevant to the study of fat biology, as energy balance is ultimately an organismal process that involves behavior, nutrient digestion, uptake and transport, as well as a variety of cellular activities that determine the balance between lipid storage and utilization. C. elegans offers the opportunity to dissect these pathways and various cellular and organismal homeostatic mechanisms in the context of a genetically tractable, intact organism.     

Structural recognition of the mRNA 3′ UTR by PUF-8 restricts the lifespan of C. elegans

The molecular mechanisms of aging are unsolved fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding with the 3′ untranslated regions (3′ UTR) in the target mRNAs. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF-1, a mitochondrial fission factor, and subsequently regulated longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. Those mutations reduced the fertility and extended the lifespan of C. elegans. Deep sequencing of total mRNAs from wild-type and puf-8 mutant worms as well as in vivo RNA Crosslinking and Immunoprecipitation (CLIP) experiments identified six PUF-8 regulated genes, which contain at least one PUF-binding element (PBE) at the 3′ UTR. One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8 mutant animals. We conclude that PUF-8 regulate the lifespan of C. elegans may not only via MFF but also via modulating pqm-1-related pathways.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

Nanoparticle imaging and diagnostic of Caenorhabditis elegans intracellular pH

A novel diagnostic tool has been developed for the characterization of intracellular pH (pHi) in the model organism Caenorhabditis elegans. This tool exploits the chemical stability of colloidal silica and the pH sensitivity of certain fluorescent dyes. Once ingested, the fluorescent colloidal dispersion yields a reliable visual indication of pH without the use of chemical fixatives or damaging the nematode. The pH-sensitive silica nanoparticles were visualized by confocal microscopy, and the fluorescence spectra from the internally referenced colloidal particulates were measured. By comparing the fluorescence profile of colloids in wild-type (N2) and mutant (eat-3) C. elegans against a calibration series, the intestinal pHi could be established in each population. The rapid characterization of pHi using this inexpensive nonintrusive technique has significant implications for disease research where C. elegans is used as a model organism.     

A worm rich in protein: Quantitative,differential, and global proteomics in Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) has been used with much success to study a number of biological processes. Although mostly known for its powerful forward and reverse genetics, work from many different groups over the past years has allowed this model organism to develop into a respectable system for proteomics studies as well. Large-scale survey studies led to improved genome annotation and to the generation of proteome catalogs, which set the stage for subsequent targeted proteomics studies. A number of focused comparative studies contributed to a better understanding of insulin signaling, spermatogenesis, oogenesis, and differential gene expression during development. In addition, C. elegans subproteomes and posttranslational modifications like glycosylation and phosphorylation have been identified. Here we describe the history of C. elegans proteomics, and provide a survey of the different methods that have been applied for relative and absolute quantification in comparative and global protein profiling studies in the worm. These studies suggest that C. elegans will provide a rich trove for “worm proteomicists”.     

An evidence-based approach to identify aging-related genes in Caenorhabditis elegans

Background

Extensive studies have been carried out on Caenorhabditis elegans as a model organism to elucidate mechanisms of aging and the effects of perturbing known aging-related genes on lifespan and behavior. This research has generated large amounts of experimental data that is increasingly difficult to integrate and analyze with existing databases and domain knowledge. To address this challenge, we demonstrate a scalable and effective approach for automatic evidence gathering and evaluation that leverages existing experimental data and literature-curated facts to identify genes involved in aging and lifespan regulation in C. elegans.

Results

We developed a semantic knowledge base for aging by integrating data about C. elegans genes from WormBase with data about 2005 human and model organism genes from GenAge and 149 genes from GenDR, and with the Bio2RDF network of linked data for the life sciences. Using HyQue (a Semantic Web tool for hypothesis-based querying and evaluation) to interrogate this knowledge base, we examined 48,231 C. elegans genes for their role in modulating lifespan and aging. HyQue identified 24 novel but well-supported candidate aging-related genes for further experimental validation.

Conclusions

We use semantic technologies to discover candidate aging genes whose effects on lifespan are not yet well understood. Our customized HyQue system, the aging research knowledge base it operates over, and HyQue evaluations of all C. elegans genes are freely available at http://hyque.semanticscience.org.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0469-4) contains supplementary material, which is available to authorized users.     

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.     

Alkyl-methylimidazolium ionic liquids affect the growth and fermentative metabolism of Clostridium sp

In this study, the effect of ionic liquids, 1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-ethyl-3-methylimidazolium diethylphosphate [EMIM][DEP], and 1-methyl-3-methylimidazolium dimethylphosphate [MMIM][DMP] on the growth and glucose fermentation of Clostridium sp. was investigated. Among the three ionic liquids tested, [MMIM][DMP] was found to be least toxic. Growth of Clostridium sp. was not inhibited up to 2.5, 4 and 4 g L⁻¹ of [EMIM][Ac], [EMIM][DEP] and [MMIM][DMP], respectively. [EMIM][Ac] at <2.5 g L⁻¹, showed hormetic effect and stimulated the growth and fermentation by modulating medium pH. Total organic acid production increased in the presence of 2.5 and 2 g L⁻¹ of [EMIM][Ac] and [MMIM][DMP]. Ionic liquids had no significant influence on alcohol production at <2.5 g L⁻¹. Total gas production was affected by ILs at ?2.5 g L⁻¹ and varied with type of methylimidazolium IL. Overall, the results show that the growth and fermentative metabolism of Clostridium sp. is not impacted by ILs at concentrations below 2.5 g L⁻¹.     

Genome-Wide RNAi Longevity Screens in Caenorhabditis elegans

Progress in aging research has identified genetic and environmental factors that regulate longevity across species. The nematode worm Caenorhabditis elegans is a genetically tractable model system that has been widely used to investigate the molecular mechanisms of aging, and the development of RNA interference (RNAi) technology has provided a powerful tool for performing large-scale genetic screens in this organism. Genome-wide screens have identified hundreds of genes that influence lifespan, many of which fall into distinct functional classes and pathways. The purpose of this review is to summarize the results of large-scale RNAi longevity screens in C. elegans, and to provide an in-depth comparison and analysis of their methodology and most significant findings.     

Something worth dyeing for: Molecular tools for the dissection of lipid metabolism in Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) has during the last decade emerged as an invaluable eukaryotic model organism to understand the metabolic and neuro-endocrine regulation of lipid accumulation. The fundamental pathways of food intake, digestion, metabolism, and signalling are evolutionary conserved between mammals and worms making C. elegans a genetically and metabolically extremely tractable model to decipher new regulatory mechanisms of lipid storage and to understand how nutritional and genetic perturbations can lead to obesity and other metabolic diseases. Besides providing an overview of the most important regulatory mechanisms of lipid accumulation in C. elegans, we also critically assess the current methodologies to monitor lipid storage and content as various methods differ in their applicability, consistency, and simplicity.     

Genetic structure and distribution of Gelidium elegans (Gelidiales,Rhodophyta) in Korea based on mitochondrial cox1 sequence data

Gelidium elegans Kützing is commonly found in Korea, China, and Japan, and is the economically most important agarophyte in the northwest Pacific. To assess the genetic structure of the Korean species, we analyzed 1200 base pairs of the mitochondrial cox1 gene from 272 individuals collected from 36 locations. A total of 34 haplotypes were found, most of which were unique, including 27 (79%) ‘private’ haplotypes. The nucleotide and haplotype diversities of cox1 within G. elegans were 0.711 ± 0.028 (H) and 0.00736 ± 0.00038 (π), respectively. The distribution of cox1 haplotypes, pairwise F_ST values, results of neutrality tests, AMOVA, and mismatch distribution revealed the existence of a deep genetic break between central Pacific Japan and all the other locations, corresponding to the surface seawater current patterns as well as the genetic signature of potential demographic expansion.     

Micelle formation in the presence of photosystem I

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (C_xG₂ with x = 10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC) = n¯₀ [prot], where the constant n¯₀ = (2.0 ± 0.3) μM⁻¹ is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x = 10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.     

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.     

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr (simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.     

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H₂O₂ are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.