Cocaine Modulates Locomotion Behavior in C. elegans

Cocaine, a potent addictive substance, is an inhibitor of monoamine transporters, including DAT (dopamine transporter), SERT (serotonin transporter) and NET (norepinephrine transporter). Cocaine administration induces complex behavioral alterations in mammals, but the underlying mechanisms are not well understood. Here, we tested the effect of cocaine on C. elegans behavior. We show for the first time that acute cocaine treatment evokes changes in C. elegans locomotor activity. Interestingly, the neurotransmitter serotonin, rather than dopamine, is required for cocaine response in C. elegans. The C. elegans SERT MOD-5 is essential for the effect of cocaine, consistent with the role of cocaine in targeting monoamine transporters. We further show that the behavioral response to cocaine is primarily mediated by the ionotropic serotonin receptor MOD-1. Thus, cocaine modulates locomotion behavior in C. elegans primarily by impinging on its serotoninergic system.     

A Monoclonal Antibody Toolkit for C. elegans

Background

Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms.

Methodology/Principal Findings

We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working in whole mount immunocytochemistry, most of these antibodies work on western blots and thus should be of use for biochemical fractionation studies.

Conclusions/Significance

We have produced a set of monoclonal antibodies to subcellular components of the nematode C. elegans for the research community. These reagents are being made available through the Developmental Studies Hybridoma Bank (DSHB).     

The Geometry of Locomotive Behavioral States in C. elegans

We develop a new hidden Markov model-based method to analyze C elegans locomotive behavior and use this method to quantitatively characterize behavioral states. In agreement with previous work, we find states corresponding to roaming, dwelling, and quiescence. However, we also find evidence for a continuum of intermediate states. We suggest that roaming, dwelling, and quiescence may best be thought of as extremes which, mixed in any proportion, define the locomotive repertoire of C elegans foraging and feeding behavior.     

A Sexually Conditioned Switch of Chemosensory Behavior in C. elegans

In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites'' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.     

3-D Worm Tracker for Freely Moving C. elegans

The manner in which the nervous system regulates animal behaviors in natural environments is a fundamental issue in biology. To address this question, C. elegans has been widely used as a model animal for the analysis of various animal behaviors. Previous behavioral assays have been limited to two-dimensional (2-D) environments, confining the worm motion to a planar substrate that does not reflect three-dimensional (3-D) natural environments such as rotting fruits or soil. Here, we develop a 3-D worm tracker (3DWT) for freely moving C. elegans in 3-D environments, based on a stereoscopic configuration. The 3DWT provides us with a quantitative trajectory, including the position and movement direction of the worm in 3-D. The 3DWT is also capable of recording and visualizing postures of the moving worm in 3-D, which are more complex than those in 2-D. Our 3DWT affords new opportunities for understanding the nervous system function that regulates animal behaviors in natural 3-D environments.     

The Influence of Bacterial Diet on Fat Storage in C. elegans

Background

The nematode Caenorhabditis elegans has emerged as an important model for studies of the regulation of fat storage. C. elegans feed on bacteria, and various strains of E. coli are commonly used in research settings. However, it is not known whether particular bacterial diets affect fat storage and metabolism.

Methodology/Principal Findings

Fat staining of fixed nematodes, as well as biochemical analysis of lipid classes, revealed considerable differences in fat stores in C. elegans growing on four different E. coli strains. Fatty acid composition and carbohydrate levels differ in the E. coli strains examined in these studies, however these nutrient differences did not appear to have a causative effect on fat storage levels in worms. Analysis of C. elegans strains carrying mutations disrupting neuroendocrine and other fat-regulatory pathways demonstrated that the intensity of Nile Red staining of live worms does not correlate well with biochemical methods of fat quantification. Several neuroendocrine pathway mutants and eating defective mutants show higher or lower fat storage levels than wild type, however, these mutants still show differences in fat stores when grown on different bacterial strains. Of all the mutants tested, only pept-1 mutants, which lack a functional intestinal peptide transporter, fail to show differential fat stores. Furthermore, fatty acid analysis of triacylglycerol stores reveals an inverse correlation between total fat stores and the levels of 15-methylpalmitic acid, derived from leucine catabolism.

Conclusions

These studies demonstrate that nutritional cues perceived in the intestine regulate fat storage levels independently of neuroendocrine cues. The involvement of peptide transport and the accumulation of a fatty acid product derived from an amino acid suggest that specific peptides or amino acids may provide nutritional signals regulating fat metabolism and fat storage levels.     

Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans

Aberrant protein aggregation is a hallmark of many age-related diseases, yet little is known about whether proteins aggregate with age in a non-disease setting. Using a systematic proteomics approach, we identified several hundred proteins that become more insoluble with age in the multicellular organism Caenorhabditis elegans. These proteins are predicted to be significantly enriched in β-sheets, which promote disease protein aggregation. Strikingly, these insoluble proteins are highly over-represented in aggregates found in human neurodegeneration. We examined several of these proteins in vivo and confirmed their propensity to aggregate with age. Different proteins aggregated in different tissues and cellular compartments. Protein insolubility and aggregation were significantly delayed or even halted by reduced insulin/IGF-1-signaling, which also slows aging. We found a significant overlap between proteins that become insoluble and proteins that influence lifespan and/or polyglutamine-repeat aggregation. Moreover, overexpressing one aggregating protein enhanced polyglutamine-repeat pathology. Together our findings indicate that widespread protein insolubility and aggregation is an inherent part of aging and that it may influence both lifespan and neurodegenerative disease.     

Bioinformatics Analysis Identify Novel OB Fold Protein Coding Genes in C. elegans

Background

The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB) fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5–25%). Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n = 46) compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n = 344) or fruit fly D. melanogaster (n = 84). Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies.

Methodology/Principal Findings

This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans.

Conclusions/Significance

This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.     

Dopamine Receptors Antagonistically Regulate Behavioral Choice between Conflicting Alternatives in C. elegans

Caenorhabditis elegans is a useful model to study the neuronal or molecular basis for behavioral choice, a specific form of decision-making. Although it has been implied that both D1-like and D2-like dopamine receptors may contribute to the control of decision-making in mammals, the genetic interactions between D1-like and D2-like dopamine receptors in regulating decision-making are still largely unclear. In the present study, we investigated the molecular control of behavioral choice between conflicting alternatives (diacetyl and Cu²⁺) by D1-like and D2-like dopamine receptors and their possible genetic interactions with C. elegans as the assay system. In the behavioral choice assay system, mutation of dop-1 gene encoding D1-like dopamine receptor resulted in the enhanced tendency to cross the Cu²⁺ barrier compared with wild-type. In contrast, mutations of dop-2 or dop-3 gene encoding D2-like dopamine receptor caused the weak tendency to cross the Cu²⁺ barrier compared with wild-type. During the control of behavioral choice, DOP-3 antagonistically regulated the function of DOP-1. The behavioral choice phenotype of dop-2; dop-1dop-3 triple mutant further confirmed the possible antagonistic function of D2-like dopamine receptor on D1-like dopamine receptor in regulating behavioral choice. The genetic assays further demonstrate that DOP-3 might act through Gα_o signaling pathway encoded by GOA-1 and EGL-10, and DOP-1 might act through Gα_q signaling pathway encoded by EGL-30 and EAT-16 to regulate the behavioral choice. DOP-1 might function in cholinergic neurons to regulate the behavioral choice, whereas DOP-3 might function in GABAergic neurons, RIC, and SIA neurons to regulate the behavioral choice. In this study, we provide the genetic evidence to indicate the antagonistic relationship between D1-like dopamine receptor and D2-like dopamine receptor in regulating the decision-making of animals. Our data will be useful for understanding the complex functions of dopamine receptors in regulating decision-making in animals.     

PAQR-2 Regulates Fatty Acid Desaturation during Cold Adaptation in C. elegans

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant.     

SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.     

Neuron-Specific Regulation of Associative Learning and Memory by MAGI-1 in C. elegans

Background

Identifying the molecular mechanisms and neural circuits that control learning and memory are major challenges in neuroscience. Mammalian MAGI/S-SCAM is a multi-PDZ domain synaptic scaffolding protein that interacts with a number of postsynaptic signaling proteins and is thereby thought to regulate synaptic plasticity [1], [2], [3].

Principal Findings

While investigating the behavioral defects of C. elegans nematodes carrying a mutation in the single MAGI ortholog magi-1, we have identified specific neurons that require MAGI-1 function for different aspects of associative learning and memory. Various sensory stimuli and a food deprivation signal are associated in RIA interneurons during learning, while additional expression of MAGI-1 in glutamatergic AVA, AVD and possibly AVE interneurons is required for efficient memory consolidation, i.e. the ability to retain the conditioned changes in behavior over time. During associative learning, MAGI-1 in RIA neurons controls in a cell non-autonomous fashion the dynamic remodeling of AVA, AVD and AVE synapses containing the ionotropic glutamate receptor (iGluR) GLR-1 [4]. During memory consolidation, however, MAGI-1 controls GLR-1 clustering in AVA and AVD interneurons cell-autonomously and depends on the ability to interact with the β-catenin HMP-2.

Significance

Together, these results indicate that different aspects of associative learning and memory in C. elegans are likely carried out by distinct subsets of interneurons. The synaptic scaffolding protein MAGI-1 plays a critical role in these processes in part by regulating the clustering of iGluRs at synapses.     

Nuclear Hormone Receptor Regulation of MicroRNAs Controls Innate Immune Responses in C. elegans

Nuclear hormone receptors respond to small molecules such as retinoids or steroids and regulate development. Signaling in the conserved p38/PMK-1 MAP kinase pathway regulates innate immunity. In this study, we show that the Caenorhabditis elegans nuclear receptor DAF-12 negatively regulates the defense against pathogens via the downstream let-7 family of microRNAs, which directly target SKN-1, a gene downstream of PMK-1. These findings identify nuclear hormone receptors as components of innate immunity that crosstalk with the p38/PMK-1 MAP kinase pathway.