Cultivation of <em>Caenorhabditis elegans</em> in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

A Smartphone-Based Imaging Method for <em>C. elegans</em> Lawn Avoidance Assay

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually ...

Yonsei University

Yonsei University Wonju College of Medicine

The beaded filament is a cytoskeletal structure that has been found only in the lens fiber cell. It includes phakosin and filensin, two divergent members of the intermediate filament family of proteins that are also unique to the fiber cell. The authors sought to determine what function the beaded filament fulfills in the lens.

Resisting the effects of aging: a function for the fiber cell beaded filament.

The lens assembles two systems of intermediated filaments-vimentin intermediate filament (IF) and highly divergent, lens-specific beaded filament (BF)-sequentially as epithelial cells differentiate into fiber cells. The goal of this study was to identify linker proteins that integrate the different lens IF into the biology of the lens fiber cells.

Periplakin interactions with lens intermediate and beaded filaments.

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca2+-calmodulin-dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.

The multiple faces of calcineurin signaling in Caenorhabditis elegans: development, behaviour and aging.

The mammalian olfactory system detects a plethora of environmental chemicals that are perceived as odors or stimulate instinctive behaviors. Studies using odorant receptor (OR) genes have provided insight into the molecular and organizational strategies underlying olfaction in mice. One important unanswered question, however, is whether these strategies are conserved in primates. To explore this question, we examined the macaque, a higher primate phylogenetically close to humans. Here we report that the organization of sensory inputs in the macaque nose resembles that in mouse in some respects, but not others. As in mouse, neurons with different ORs are interspersed in the macaque nose, and there are spatial zones that differ in their complement of ORs and extend axons to different domains in the olfactory bulb of the brain. However, whereas the mouse has multiple discrete band-like zones, the macaque appears to have only two broad zones. It is unclear whether the organization of OR inputs in a rodent/primate common ancestor degenerated in primates or, alternatively became more sophisticated in rodents. The mouse nose has an additional small family of chemosensory receptors, called trace amine-associated receptors (TAARs), which may detect social cues. Here we find that TAARs are also expressed in the macaque nose, suggesting that TAARs may also play a role in human olfactory perception. We further find that one human TAAR responds to rotten fish, suggesting a possible role as a sentinel to discourage ingestion of food harboring pathogenic microorganisms.

Olfactory receptor patterning in a higher primate.

The olfactory system translates a vast array of volatile chemicals into diverse odor perceptions and innate behaviors. Odor detection in the mouse nose is mediated by 1,000 different odorant receptors (ORs) and 14 trace amine-associated receptors (TAARs). ORs are used in a combinatorial manner to encode the unique identities of myriad odorants. However, some TAARs appear to be linked to innate responses, raising questions about regulatory mechanisms that might segregate OR and TAAR expression in appropriate subsets of olfactory sensory neurons (OSNs). Here, we report that OSNs that express TAARs comprise at least two subsets that are biased to express TAARs rather than ORs. The two subsets are further biased in Taar gene choice and their distribution within the sensory epithelium, with each subset preferentially expressing a subgroup of Taar genes within a particular spatial domain in the epithelium. Our studies reveal one mechanism that may regulate the segregation of Olfr (OR) and Taar expression in different OSNs: the sequestration of Olfr and Taar genes in different nuclear compartments. Although most Olfr genes colocalize near large central heterochromatin aggregates in the OSN nucleus, Taar genes are located primarily at the nuclear periphery, coincident with a thin rim of heterochromatin. Taar-expressing OSNs show a shift of one Taar allele away from the nuclear periphery. Furthermore, examination of hemizygous mice with a single Taar allele suggests that the activation of a Taar gene is accompanied by an escape from the peripheral repressive heterochromatin environment to a more permissive interior chromatin environment.

Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments.

Animal predators can track prey using their keen sense of smell. The bacteriovorous nematode Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like odor receptors to locate bacteria. C. elegans displays odor-related behaviors such as attraction, aversion and adaptation, but the ecological significance of these behaviors is not known. Using a combination of food microbiology and genetics, we elucidate a possible predator-prey relationship between C. elegans and lactic acid bacteria (LAB) in rotting citrus fruit. LAB produces the volatile odor diacetyl as an oxidized by-product of fermentation in the presence of citrate. We show that C. elegans is attracted to LAB when grown on citrate media or Citrus medica L, commonly known as yuzu, a citrus fruit native to East Asia, and this attraction is mediated by the diacetyl odor receptor, ODR-10. We isolated a wild LAB strain and a wild C. elegans-related nematode from rotten yuzu, and demonstrate that the wild nematode was attracted to the diacetyl produced by LAB. These results not only identify an ecological function for a C. elegans olfactory behavior, but contribute to the growing understanding of ecological relationships between the microbial and metazoan worlds.

A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.

Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein's function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains.

Violacein: Properties and Production of a Versatile Bacterial Pigment.

The nematodeCaenorhabditiselegansis one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a two dimensional habitat. In nature, however,C. elegansis found in three dimensional environments such as rotting fruit. To investigate the biology ofC. elegansin a 3D controlled environment we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. NGT-3D allows for the growth of both nematodes and the bacteria they consume. Worms show comparable rates of growth, reproduction and lifespan when bacterial colonies in the 3D matrix are abundant. However, when bacteria are sparse, growth and brood size fail to reach levels observed in standard 2D plates. Using NGT-3D we observe drastic deficits in fertility in a sensory mutant in 3D compared to 2D, and this defect was likely due to an inability to locate bacteria. Overall, NGT-3D will sharpen our understanding of nematode biology and allow scientists to investigate questions of nematode ecology and evolutionary fitness in the laboratory.

NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.

The mechanisms by which odors induce instinctive behaviors are largely unknown. Odor detection in the mouse nose is mediated by >1, 000 different odorant receptors (ORs) and trace amine-associated receptors (TAARs). Odor perceptions are encoded combinatorially by ORs and can be altered by slight changes in the combination of activated receptors. However, the stereotyped nature of instinctive odor responses suggests the involvement of specific receptors and genetically programmed neural circuits relatively immune to extraneous odor stimuli and receptor inputs. Here, we report that, contrary to expectation, innate odor-induced behaviors can be context-dependent. First, different ligands for a given TAAR can vary in behavioral effect. Second, when combined, some attractive and aversive odorants neutralize one another's behavioral effects. Both a TAAR ligand and a common odorant block aversion to a predator odor, indicating that this ability is not unique to TAARs and can extend to an aversive response of potential importance to survival. In vitro testing of single receptors with binary odorant mixtures indicates that behavioral blocking can occur without receptor antagonism in the nose. Moreover, genetic ablation of a single receptor prevents its cognate ligand from blocking predator odor aversion, indicating that the blocking requires sensory input from the receptor. Together, these findings indicate that innate odor-induced behaviors can depend on context, that signals from a single receptor can block innate odor aversion, and that instinctive behavioral responses to odors can be modulated by interactions in the brain among signals derived from different receptors.

Combinatorial effects of odorants on mouse behavior.

As space flight becomes more accessible in the future, humans will be exposed to gravity conditions other than our 1G environment on Earth. Our bodies and physiology, however, are adapted for life at 1G gravity. Altering gravity can have profound effects on the body, particularly the development of muscles, but the reasons and biology behind gravity's effect are not fully known. We asked whether increasing gravity had effects on the development of motor neurons that innervate and control muscle, a relatively unexplored area of gravity biology. Using the nematode model organism Caenorhabditis elegans, we examined changes in response to hypergravity in the development of the 19 GABAergic DD/VD motor neurons that innervate body muscle. We found that a high gravity force above 10G significantly increases the number of animals with defects in the development of axonal projections from the DD/VD neurons. We showed that a critical period of hypergravity exposure during the embryonic/early larval stage was sufficient to induce defects. While characterizing the nature of the axonal defects, we found that in normal 1G gravity conditions, DD/VD axonal defects occasionally occurred, with the majority of defects occurring on the dorsal side of the animal and in the mid-body region, and a significantly higher rate of error in the 13 VD axons than the 6 DD axons. Hypergravity exposure increased the rate of DD/VD axonal defects, but did not change the distribution or the characteristics of the defects. Our study demonstrates that altering gravity can impact motor neuron development.

Hypergravity hinders axonal development of motor neurons in Caenorhabditis elegans.

Maternal behaviors benefit the survival of young, contributing directly to the mother's reproductive fitness. An extreme form of this is seen in matriphagy, when a mother performs the ultimate sacrifice and offers her body as a meal for her young. Whether matriphagy offers only a single energy-rich meal or another possible benefit to the young is unknown. Here, we characterized the toxicity of a bacterial secondary metabolite, namely, violacein, in Caenorhabditis elegans and found it is not only toxic towards adults, but also arrests growth and development of C. elegans larvae. To counteract this, C. elegans resorted to matriphagy, with the mothers holding their eggs within their bodies and hatching the young larvae internally, which eventually led to the mothers' death. This violacein-induced matriphagy alleviated some of the toxic effects of violacein, allowing a portion of the internally-hatched young to bypass developmental arrest. Using genetic and pharmacological experiments, we found the consumption of oleate, a monounsaturated fatty acid produced by the mother, during matriphagy is partially responsible. As such, our study provides experimental evidence of why such a drastic and peculiar maternal behavior may have arisen in nematode natural habitats.

Consumption of Oleic Acid During Matriphagy in Free-Living Nematodes Alleviates the Toxic Effects of the Bacterial Metabolite Violacein.

Biology is adapted to Earth's gravity force, and the long-term effects of varying gravity on the development of animals is unclear. Previously, we reported that high gravity, called hypergravity, increases defects in the development of motor neuron axons in the nematode Caenorhabditis elegans. Here, we show that a mutation in the unc-70 gene that encodes the cytoskeletal β-spectrin protein suppresses hypergravity-induced axon defects. UNC-70 expression is required in both muscle and epidermis to promote the axon defects in high gravity. We reveal that the location of axon defects is correlated to the size of the muscle cell that the axon traverses. We also show that mutations that compromise key proteins of hemidesmosomal structures suppress hypergravity-induced axon defects. These hemidesmosomal structures play a crucial role in coupling mechanical force between the muscle, epidermis and the external cuticle. We speculate a model in which the rigid organization of muscle, epidermal and cuticular layers under high gravity pressure compresses the narrow axon migration pathways in the extracellular matrix hindering proper axon pathfinding of motor neurons.

Muscle and epidermal contributions of the structural protein β-spectrin promote hypergravity-induced motor neuron axon defects in C. elegans.

In this review, we discuss violacein and prodigiosin, two chromogenic bacterial secondary metabolites that have diverse biological activities. Although both compounds were "discovered" more than seven decades ago, interest into their biological applications has grown in the last two decades, particularly driven by their antimicrobial and anticancer properties. These topics will be discussed in the first half of this review. The latter half delves into the current efforts of groups to produce these two compounds. This includes in both their native bacterial hosts and heterogeneously in other bacterial hosts, including discussing some of the caveats related to the yields reported in the literature, and some of the synthetic biology techniques employed in this pursuit.

Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin.

Our knowledge of animal and behavior in the natural ecology is based on over a century's worth of valuable field studies. In this post-genome era, however, we recognize that genes are the underpinning of ecological interactions between two organisms. Understanding how genes contribute to animal ecology, which is essentially the intersection of two genomes, is a tremendous challenge. The bacterivorous nematode , one of the most well-known genetic animal model experimental systems, experiences a complex microbial world in its natural habitat, providing us with a window into the interplay of genes and molecules that result in an animal-microbial ecology. In this review, we will discuss  natural ecology, how the worm uses its sensory system to detect the microbes and metabolites that it encounters, and then discuss some of the fascinating ecological dances, including behaviors, that have evolved between the nematode and the microbes in its environment.

Kyoung-hye Yoon

Division of Biological Science and Technology

Yonsei University

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

Kyoung-hye Yoon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Biological Science and Technology

Yonsei University

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

Division of Biological Science and Technology