An improved solid medium-based culturing method for Steinernema hermaphroditum.

Steinernema hermaphroditum is the only identified entomopathogenic nematode that is consistently hermaphroditic and thus offers a great opportunity to use genetic approaches to probe symbiosis. Evolutionarily, ecologically, and morphologically distinct from laboratory nematodes commonly used in the laboratory, with both forward and reverse genetics tools available, this species also provides an opportunity to explore other areas of biology, especially using comparative studies. Here, we describe an improved solid medium-based culturing method for S. hermaphroditum that we found particularly helpful for phenotypic analysis and genetic manipulation. We document the rapid increase in the size of the worm; and show that the uniform growth of the worm on this medium provides a good basis for developmental studies. Finally, we measure the brood size of individual animals, which, although far larger, has a very similar trajectory to that of the hermaphroditic Caenorhabditis elegans, suggesting common reproductive restraints.

Lysosome-related organelles contain an expansion compartment that mediates delivery of zinc transporters to promote homeostasis.

Zinc is an essential nutrient-it is stored during periods of excess to promote detoxification and released during periods of deficiency to sustain function. Lysosome-related organelles (LROs) are an evolutionarily conserved site of zinc storage, but mechanisms that control the directional zinc flow necessary for homeostasis are not well understood. In Caenorhabditis elegans intestinal cells, the CDF-2 transporter stores zinc in LROs during excess. Here, we identify ZIPT-2.3 as the transporter that releases zinc during deficiency; ZIPT-2.3 transports zinc, localizes to the membrane of LROs in intestinal cells, and is necessary for zinc release from LROs and survival during zinc deficiency. In zinc excess and deficiency, the expression levels of CDF-2 and ZIPT-2.3 are reciprocally regulated at the level of mRNA and protein, establishing a fundamental mechanism for directional flow to promote homeostasis. To elucidate how the ratio of CDF-2 and ZIPT-2.3 is altered, we used super-resolution microscopy to demonstrate that LROs are composed of a spherical acidified compartment and a hemispherical expansion compartment. The expansion compartment increases in volume during zinc excess and deficiency. These results identify the expansion compartment as an unexpected structural feature of LROs that facilitates rapid transitions in the composition of zinc transporters to mediate homeostasis, likely minimizing the disturbance to the acidified compartment.

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematode Steinernema hermaphroditum.

The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism of its insect hosts and mutualism with its bacterial endosymbiont Xenorhabdus griffiniae. Through whole-genome re-sequencing and genetic mapping we have for the first time molecularly identified the gene responsible for a mutationally defined phenotypic locus in an entomopathogenic nematode. In the process we observed an unexpected mutational spectrum following ethyl methansulfonate mutagenesis in this species. We find that the ortholog of the essential Caenorhabditis elegans peroxidase gene skpo-2 controls body size and shape in S. hermaphroditum. We confirmed this identification by generating additional loss-of-function mutations in the gene using CRISPR-Cas9. We propose that the identification of skpo-2 will accelerate gene targeting in other Steinernema entomopathogenic nematodes used commercially in pest control, as skpo-2 is X-linked and males hemizygous for loss of its function can mate, making skpo-2 an easily recognized and maintained marker for use in co-CRISPR.

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans.

The biological roles of the autofluorescent lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, have been shown to play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules increased and then decreased in a rapid and dynamic manner that may be associated with nutrient availability. We observed that two distinct types of fluorophores are likely present in the gut granules. One displays a "flashing" phenomenon, in which fluorescence decrease is preceded by a sharp increase in fluorescence intensity that expands into the surrounding area, while the other simply decreases in intensity. Gut granule flashing was observed in the different life stages of C. elegans and was also observed in Steinernema hermaphroditum, an evolutionarily distant nematode. We hypothesize that the "flashing" fluorophore is pH-sensitive, and the fluorescence intensity change results from the fluorophore being released from the lysosome-related organelles into the relatively higher pH environment of the cytosol. The visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on the biochemistry and genetics of these lysosome-related organelles.

LINKIN-associated proteins necessary for tissue integrity during collective cell migration.

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1, Caenorhabditis elegans: lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. In C. elegans, loss of lnkn-1 results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1 - RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin - were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1. Loss-of-function analysis of their C. elegans orthologs found that three of the interactors - ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2 - displayed migratory detachment phenotypes similar to that of lnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that of lnkn-1 (notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.

Loss of famh-136/ FAM136A results in minor locomotion and behavioral changes in Caenorhabditis elegans.

Not much is known about FAM136A, a human gene that may be involved in Meniere's disease and is conserved throughout animals. To understand the function of famh-136 , the Caenorhabditis elegans ortholog of FAM136A, loss of function alleles of the gene were generated. We find that loss of famh-136 function results in minor but significant changes to the locomotion and behavior.

DiI staining of sensory neurons in the entomopathogenic nematode Steinernema hermaphroditum.

Steinernema hermaphroditum entomopathogenic nematodes (EPN) and their Xenorhabdus griffiniae symbiotic bacteria have recently been shown to be a genetically tractable system for the study of both parasitic and mutualistic symbiosis. In their infective juvenile (IJ) stage, EPNs search for insect hosts to invade and quickly kill them with the help of the symbiotic bacteria they contain. The mechanisms behind these behaviors have not been well characterized, including how the nematodes sense their insect hosts. In the well-studied free­living soil nematode Caenorhabditis elegans, ciliated amphid neurons enable the worms to sense their environment, including chemosensation. Some of these neurons have also been shown to control the decision to develop as a stress-resistant dauer larva, analogous to the infective juveniles of EPNs, or to exit from dauer and resume larval development. In C. elegans and other nematodes, dye-filling with DiI is an easy and efficient method to label these neurons. We developed a protocol for DiI staining of S. hermaphroditum sensory neurons. Using this method, we could identify neurons positionally analogous to the C. elegans amphid neurons ASI, ADL, ASK, ASJ, as well as inner labial neurons IL1 and IL2. Similar to findings in other EPNs, we also found that the IJs of S. hermaphroditum are dye-filling resistant.

A laboratory and simulation platform to integrate individual life history traits and population dynamics.

Understanding populations is important because they are a fundamental level of biological organization. Individual traits such as aging and lifespan interact in complex ways to determine birth and death and thereby influence population dynamics. However, we lack a deep understanding of the relationships between individual traits and population dynamics. To address this challenge, we established a laboratory population using the model organism C. elegans and an individual-based computational simulation informed by measurements of real worms. The simulation realistically models individual worms and the behavior of the laboratory population. To elucidate the role of aging in population dynamics, we analyzed old age as a cause of death and showed, using computer simulations, that it was influenced by maximum lifespan, rate of adult culling, and progeny number/food stability. Notably, populations displayed a tipping point for aging as the primary cause of adult death. Our work establishes a conceptual framework that could be used for better understanding why certain animals die of old age in the wild.

The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis.

Entomopathogenic nematodes (EPNs), including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in EPNs has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as producing hermaphrodites in the first generation. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic EPN and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

Zinc is an intracellular signal during sperm activation in Caenorhabditis elegans.

Sperm activation is a rapid and dramatic cell differentiation event that does not involve changes in transcription, and the signaling cascades that mediate this process have not been fully defined. zipt-7.1 encodes a zinc transporter, and zipt-7.1(lf) mutants display sperm-activation defects, leading to the hypothesis that zinc signaling mediates sperm activation in Caenorhabditis elegans. Here, we describe the development of a method for dynamic imaging of labile zinc during sperm activation using the zinc-specific fluorescence probe FluoZin-3 AM and time-lapse confocal imaging. Two phases of dynamic changes in labile zinc levels were observed during sperm activation. Forced zinc entry using the zinc ionophore pyrithione activated sperm in vitro, and it suppressed the defects of zipt-7.1(lf) mutants, indicating that high levels of cytosolic zinc are sufficient for sperm activation. We compared activation by zinc pyrithione to activation by extracellular zinc, the Na+/H+ antiporter monensin and the protease cocktail pronase in multiple mutant backgrounds. These results indicate that the protease pathway does not require zinc signaling, suggesting that zinc signaling is sufficient to activate sperm but is not always necessary.

Zinc homeostasis and signaling in the roundworm C. elegans.

C. elegans is a powerful model for studies of zinc biology. Here we review recent discoveries and emphasize the advantages of this model organism. Methods for manipulating and measuring zinc levels have been developed in or adapted to the worm. The C. elegans genome encodes highly conserved zinc transporters, and their expression and function are beginning to be characterized. Homeostatic mechanisms have evolved to respond to high and low zinc conditions. The pathway for high zinc homeostasis has been recently elucidated based on the discovery of the master regulator of high zinc homeostasis, HIZR-1. A parallel pathway for low zinc homeostasis is beginning to emerge based on the discovery of the Low Zinc Activation promoter element. Zinc has been established to play a role in two cell fate determination events, and accumulating evidence suggests zinc may function as a second messenger signaling molecule during vulval cell development and sperm activation.

The zinc transporter ZIPT-7.1 regulates sperm activation in nematodes.

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process.

The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc.

Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors.

Insights into zinc and cadmium biology in the nematode Caenorhabditis elegans.

Zinc is an essential metal that is involved in a wide range of biological processes, and aberrant zinc homeostasis is implicated in multiple human diseases. Cadmium is chemically similar to zinc, but it is a nonessential environmental pollutant. Because zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to maintain zinc levels in response to dietary fluctuations. The nematode Caenorhabditis elegans is emerging as a powerful model system to investigate zinc trafficking and homeostasis as well as cadmium toxicity. Here we review genetic and molecular studies that have combined to generate a picture of zinc homeostasis based on the transcriptional control of zinc transporters in intestinal cells. Furthermore, we summarize studies of cadmium toxicity that reveal intriguing parallels with zinc biology.

C. elegans EIF-3.K promotes programmed cell death through CED-3 caspase.

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.