Conservation rules, their breakdown, and optimality in Caenorhabditis sinusoidal locomotion.

Undulatory locomotion is common to nematodes as well as to limbless vertebrates, but its control is not understood in spite of the identification of hundred of genes involved in Caenorhabditis elegans locomotion. To reveal the mechanisms of nematode undulatory locomotion, we quantitatively analysed the movement of C. elegans with genetic perturbations to neurons, muscles, and skeleton (cuticle). We also compared locomotion of different Caenorhabditis species. We constructed a theoretical model that combines mechanics and biophysics, and that is constrained by the observations of propulsion and muscular velocities, as well as wavelength and amplitude of undulations. We find that normalized wavelength is a conserved quantity among wild-type C. elegans individuals, across mutants, and across different species. The velocity of forward propulsion scales linearly with the velocity of the muscular wave and the corresponding slope is also a conserved quantity and almost optimal; the exceptions are in some mutants affecting cuticle structure. In theoretical terms, the optimality of the slope is equivalent to the exact balance between muscular and visco-elastic body reaction bending moments. We find that the amplitude and frequency of undulations are inversely correlated and provide a theoretical explanation for this fact. These experimental results are valid both for young adults and for all larval stages of wild-type C. elegans. In particular, during development, the amplitude scales linearly with the wavelength, consistent with our theory. We also investigated the influence of substrate firmness on motion parameters, and found that it does not affect the above invariants. In general, our biomechanical model can explain the observed robustness of the mechanisms controlling nematode undulatory locomotion.

Heterotrimeric G proteins in C. elegans.

Heterotrimeric G proteins, composed of alpha, beta, and gamma subunits, are able to transduce signals from membrane receptors to a wide variety of intracellular effectors. In this role, G proteins effectively function as dimers since the signal is communicated either by the G alpha subunit or the stable G betagamma complex. When inactive, G alpha-GDP associates with G betagamma and the cytoplasmic portion of the receptor. Ligand activation of the receptor stimulates an exchange of GTP for GDP resulting in the active signaling molecules G alpha-GTP and free G betagamma, either of which can interact with effectors. Hydrolysis of GTP restores G alpha-GDP, which then reassociates with G betagamma and receptor to terminate signaling. The rate of G protein activation can be enhanced by the guanine-nucleotide exchange factor, RIC-8, while the rate of GTP hydrolysis can be enhanced by RGS proteins such as EGL-10 and EAT-16. Evidence for a receptor-independent G-protein-signaling pathway has been demonstrated in C. elegans early embryogenesis. In this pathway, the G alpha subunits GOA-1 and GPA-16 are apparently activated by the non-transmembrane proteins GPR-1, GPR-2, and RIC-8, and negatively regulated by RGS-7. The C. elegans genome encodes 21 G alpha, 2 G beta and 2 G gamma subunits. The alpha subunits include one ortholog of each mammalian G alpha family: GSA-1 (Gs), GOA-1 (Gi/o), EGL-30 (Gq) and GPA-12 (G12). The remaining C. elegans alpha subunits (GPA-1, GPA-2, GPA-3, GPA-4, GPA-5, GPA-6, GPA-7, GPA-8, GPA-9, GPA-10, GPA-11, GPA-13, GPA-14, GPA-15, GPA-16, GPA-17 and ODR-3) are most similar to the Gi/o family, but do not share sufficient homology to allow classification. The conserved G alpha subunits, with the exception of GPA-12, are expressed broadly while 14 of the new G alpha genes are expressed in subsets of chemosensory neurons. Consistent with their expression patterns, the conserved C. elegans alpha subunits, GSA-1, GOA-1 and EGL-30 are involved in diverse and fundamental aspects of development and behavior. GOA-1 acts redundantly with GPA-16 in positioning of the mitotic spindle in early embryos. EGL-30 and GSA-1 are required for viability starting from the first larval stage. In addition to their roles in development and behaviors such as egg laying and locomotion, the EGL-30, GSA-1 and GOA-1 pathways interact in a network to regulate acetylcholine release by the ventral cord motor neurons. EGL-30 provides the core signals for vesicle release, GOA-1 negatively regulates the EGL-30 pathway, and GSA-1 modulates this pathway, perhaps by providing positional cues. Constitutively activated GPA-12 affects pharyngeal pumping. The G alpha subunits unique to C. elegans are primarily involved in chemosensation. The G beta subunit, GPB-1, as well as the G gamma subunit, GPC-2, appear to function along with the alpha subunits in the classic G protein heterotrimer. The remaining G beta subunit, GPB-2, is thought to regulate the function of certain RGS proteins, while the remaining G gamma subunit, GPC-1, has a restricted role in chemosensation. The functional difference for most G protein pathways in C. elegans, therefore, resides in the alpha subunit. Many cells in C. elegans express multiple G alpha subunits, and multiple G protein pathways are known to function in specific cell types. For example, Go, Gq and Gs-mediated signaling occurs in the ventral cord motor neurons. Similarly, certain amphid neurons use multiple G protein pathways to both positively and negatively regulate chemosensation. C. elegans thus provides a powerful model for the study of interactions between and regulation of G protein signaling.

Regulation of sphingosine-1-phosphate lyase gene expression by members of the GATA family of transcription factors.

Sphingosine-1-phosphate is a bioactive sphingolipid that regulates proliferation, differentiation, migration, and apoptosis. Sphingosine-1-phosphate is irreversibly degraded by the highly conserved enzyme sphingosine-1-phosphate lyase. Recent studies have suggested that sphingosine-1-phosphate lyase expression affects animal development and cell fate decisions. Despite its crucial role, mechanisms affecting expression of sphingosine-1-phosphate lyase remain poorly understood. In this study, regulation of sphingosine-1-phosphate lyase gene expression was investigated in Caenorhabditis elegans, where lyase expression is spatially restricted to cells of the developing and adult gut and is essential for normal development. Deletion analysis and generation of transgenic worms combined with fluorescence microscopy identified a 350-nucleotide sequence upstream of the ATG start site necessary for maximal lyase expression in adult worms. Site-specific mutagenesis of a GATA transcription factor-binding motif in the promoter led to loss of reporter expression. Knockdown of the gut-specific GATA transcription factor ELT-2 by RNA interference similarly led to loss of reporter expression. ELT-2 interacted with the GATA factor-binding motif in vitro and was also capable of driving expression of a Caenorhabditis elegans lyase promoter-beta-galactosidase reporter in a heterologous yeast system. These studies demonstrate that ELT-2 regulates sphingosine-1-phosphate lyase expression in vivo. Additionally, we demonstrate that the human sphingosine-1-phosphate lyase gene is regulated by a GATA transcription factor. Overexpression of GATA-4 led to both an increase in activity of a reporter gene as well as an increase in endogenous sphingosine-1-phosphate lyase protein.

An automated system for measuring parameters of nematode sinusoidal movement.

BACKGROUND: Nematode sinusoidal movement has been used as a phenotype in many studies of C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior. RESULTS: We have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worm's centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements. To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly (activation of Gq-alpha versus loss of Go-alpha function), as well as of different mutant alleles at a single locus (null and dominant negative alleles of the goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb. CONCLUSIONS: Automated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans.

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.