Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

Cholinergic system plays important role in all functions of organisms of free-living soil nematodes C. elegans and C. briggsae. Using pharmacological analysis we showed the existence of two opposite responses of nematodes cholinergic system to moderate and extreme heat stress. Short-term (15min) noxious heat (31-32°C) caused activation of cholinergic synaptic transmission in C. elegans and C. briggsae organisms by sensitization of nicotinic ACh receptors. In contrast, hyperthermia blocked cholinergic synaptic transmission by inhibition of ACh secretion by neurons. The resistance of behavior to extreme high temperature (36-37°C) was significantly higher in C. briggsae than in C. elegans, and thermostability of cholinergic transmission correlated with resistance of behavior to hyperthermia. Activation of cholinergic transmission by moderate heat stress can be the reason of movement speed increase in such adaptive behavior as noxious heat escape. Inhibition of ACh release is one of reasons for behavior failure caused by extreme high temperature since partial inhibition of ACh-esterase by aldicarb protected C. elegans and C. briggsae behavior against hyperthermia. Antagonist of mAChRs atropine almost completely prevented the rise in behavior thermotolerance caused by aldicarb. Pilocarpine, agonist of mAChRs, protected nematodes behavior against hyperthermia similarly with aldicarb. Therefore it is evident that it is the deficiency of mAChRs activity that is the reason for nematodes' behavior failure by hyperthermia.

HandKAchip - Hands-free killing assay on a chip.

Small animals such as the roundworm C. elegans are excellent models for studying bacterial infection and host response, as well as for genetic and chemical screens. A key methodology is the killing assay, in which the number of surviving animals is tracked as a function of the time post infection. This is a labor-intensive procedure, prone to human error and subjective choices, and often involves undesired perturbation to the animals and their environment. In addition, the survival of animals is just one aspect of a multi-dimensional complex biological process. Here we report a microfluidic-based approach for performing killing assays in worms, compatible with standard assays performed on solid media. In addition to providing accurate and reproducible survival curves at a considerably reduced labor, this approach allows acquisition of a multitude of quantitative data with minimal undesired perturbations. These measurements are obtained automatically at a worm-by-worm resolution using a custom image processing workflow. The proposed approach is simple, scalable, and extendable, and is significantly more economical than standard manual protocols.

Selective conservation of the RSL-encoding, proteinase inhibitory-type, clade L serpins in Caenorhabditis species.

Serpins are a highly conserved superfamily of serine and papain-like cysteine proteinase inhibitors that are divided phylogenetically into clades. Serpins also can be divided anatomically into those that reside predominately outside or inside cells. While the activities of the extracellular serpins are well understood, the biological functions, as well as the overall distribution of the intracellular (serpinIC) serpins is less well defined. Conceivably, the biological function of the serpinsIC might be revealed by analysis of species with genomes of lower complexity. To this end, we sought to define the clade L serpin repertoire of Caenorhabditis elegans and other nematode species. Analysis of the C. elegans genome revealed the presence of 9 serpin genes. Five genes encoded for full-length serpins with functional reactive site loops (RSL). By definition, these genes were designated proteinase inhibitory-type, RSL-encoding serpins. Four of the C. elegans genes encoded for proteins without an RSL or transcripts with premature termination codons. The high percentage of non-RSL encoding to RSL-encoding serpin genes suggested that the former served a unique biological function rather than residing in the genome as simple pseudogenes. If this hypothesis was correct, we expected these non-RSL encoding genes to be conserved precisely in other Caenorhabditis species. However, in contrast to the RSL-encoding serpins that were well conserved and segregated into 3 sub-clades, we failed to detect non-RSL encoding serpin orthologues in the genomes of Caenorhabditis briggsae and Caenorhabditis remanei. These data suggested that unlike their RSL-encoding paralogues, the relatively high percentage of non-RSL encoding serpins in C. elegans was a vestige of recent duplication events and these latter genes were unlikely to serve essential functions in Caenorhabditis species.

Modelling human diseases in Drosophila and Caenorhabditis.

Drosophila (fruitfly) and Caenorhabditis (nematode worm) are useful model organisms for understanding many molecular and cellular mechanisms of human disease. Work on them is powered by versatile gene discovery methods, output of their genome projects, and a biology that has much in common with that of humans. They contain homologues of most human disease genes. Many aspects of human disease, and of defences against disease, are also found in flies and worms. These include cancer, ageing, neurodegeneration, infectious disease, innate immunity, and dysfunction of neurotransmitter and endocrine systems.

Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell.

The excretory cell extends a tubular process, or canal, along the basolateral surface of the epidermis to form the nematode renal epithelium. This cell can undergo normal tubulogenesis in isolated cell culture. Mutations in 12 genes cause excretory canal cysts in Caenorhabditis elegans. Genetic interactions, and their similar phenotypes, suggest these genes may encode functionally related proteins. Depending upon genotype and individual canal, defects range from focal cysts, flanked by normal width segments, to regional cysts involving the entire tubule. Oftentimes the enlarged regions are convoluted or partially septated. In mutants with very large cysts, renal function is measurably impaired. Based on histology and ultrastructure, canal cysts likely result from defects of the apical membrane domain. These mutants provide a model of tubulocystic disease without hyperplasia or basement membrane abnormalities. Similar apical mechanisms could regulate tubular morphology of vertebrate nephrons.

A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans.

Neurotoxicity through abnormal activation of membrane channels is a potential cause of neurodegenerative disease. Here we show that a gain-of-function mutation, deg.3(u662), leads to the degeneration of a small set of neurons in the nematode C. elegans. The deg.3 gene encodes a nicotinic acetylcholine receptor alpha subunit, which in the region of transmembrane domain II is most similar to the neuronal alpha 7 subunits from rat and chicken. The u662 mutation changes a residue in the second transmembrane domain, the domain thought to form the channel pore. A similar change in the equivalent amino acid in the chick protein produces channels that desensitize slowly. Channel hyperactivity may underlie the degenerations seen in the C. elegans deg.3(u662) mutants, since antagonists of nicotinic acetylcholine receptors suppress the deg-3(u662) mutant phenotypes.

The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons.

Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively.

Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.

The functions of the 11 classes of exposed chemosensory neurons of C. elegans were tested by killing cells with a laser microbeam. One pair of neurons, the ASE neurons, is uniquely important for chemotaxis: killing the ASE neurons greatly reduced chemotaxis to cAMP, biotin, Cl-, and Na+. Additional chemosensory function is distributed among several other cell types. Thus, 3 pairs of chemosensory neurons (ADF, ASG, and ASI) contribute to a residual response to cAMP, biotin, Cl-, and Na+ after ASE is killed. Chemotaxis to lysine similarly depends on the partly redundant functions of 4 pairs of chemosensory neurons (ASE, ASG, ASI, and ASK). The combined activity of several neuron types that act in parallel might increase the fidelity of chemotaxis.

Muscle cell attachment in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.

Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction.

The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions.

Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans.

unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.

Radiation sensitivity and DNA repair in Caenorhabditis elegans strains with different mean life spans.

The sensitivities to three DNA damaging agents (UV and gamma-radiation, methyl methanesulfonate) were measured in four recombinant inbred (RI) strains of Caenorhabditis elegans with mean life spans ranging from 13 to 30.9 days, as well as in the wild-type strains used to derive these RI's. Sensitivities at several stages in the developmental cycle were tested. There were no significant correlations between mean life span and the lethal effects of these 3 agents. Excision of two UV-radiation-induced DNA photoproducts was also measured. Long-lived strains were no more repair competent than shorter-lived strains. These data indicate that DNA repair plays at best a minor role in the aging process of C. elegans.

Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans.

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.

Caenorhabditis elegans: comparisons of chemotactic behavior from monoxenic and axenic culture.

Significant differences in chemotactic response of Caenorhabditis elegans were demonstrated for nematodes from monoxenic culture as compared to nematodes from axenic culture. These results support those of a previous study in which large differences in growth, development, behavior, and longevity were shown for C. elegans in comparative assays of the monoxenic and axenic regimes.

Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans.

Tannic acid fixation reveals differences in the number of protofilaments between microtubules (MTs) in the nematode Caenorhabditis elegans. Most cells have MTs with 11 protofilaments but the six touch receptor neurons (the microtubule cells) have MTs with 15 protofilaments. No 13-protofilament (13-p) MT has been seen. The modified cilia of sensory neurons also possess unusual structures. The cilia contain nine outer doublets with A subfibers of 13 protofilaments and B subfibers of 11 protofilaments and a variable number of inner singlet MTs containing 11 protofilaments. The 15-p MTs but not the 11-p MTs are eliminated by colchicine-treatment or by mutation of the gene mec-7. Concomitantly, touch sensitivity is also lost. However, whereas colchicine treatment leads to the loss of all MTs from the microtubule cells, mutations in mec-7 result in the partial replacement of the 15-p MTs with 11-p MTs. Benzimidazoles (benomyl and nocodazole) have more general effects on C. elegans (slow growth, severe uncoordination, and loss of processes from the ventral cord) but do not affect the 15-p MTs. Benomyl will, however, disrupt the replacement 11-p MTs found in the microtubule cells of mec-7 mutants. The 11-p and 15-p MTs also respond differently to temperature and fixation conditions. It is likely that either type of MT will suffice for the proper outgrowth of the microtubule cell process, but only the 15-p MT can function in the specialized role of sensory transduction of the microtubule cells.

Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.

The pseudopods of Caenorhabditis elegans spermatozoa move actively causing some cells to translocate when the sperm are dissected into a low osmotic strength buffered salts solution. On time-lapse video tapes, pseudopodial projections can be seen moving at 20-45 micrometers/min from the tip to the base of the pseudopod. This movement occurs whether or not the cell is attached to a substrate. Translocation of the cell is dependent on the substrate. Some spermatozoa translocate on acid-washed glass, but a better substrate is prepared by drying an extract of Ascaris uteri (the normal site of nematode sperm motility) onto glass slides. On this substrate more than half the spermatozoa translocate at a velocity (21 micrometers/min) similar to that observed in vivo. Translocating cells attach to the substrate by their pseudopodial projections. They always move toward the pseudopod; changes in direction are caused by changes in pseudopod shape that determine points of detachment and reattachment of the cell to the substrate. Actin comprises less than 0.02% of the proteins in sperm, and myosin is undetectable. No microfilaments are found in the sperm. Immunohistochemistry shows that some actin is localized in patches in the pseudopod. The movement of spermatozoa is unaffected by cytochalasins, however, so there is no evidence that actin participates in locomotion. Fertilization-defective mutants in genes fer-2, fer-4, and fer-6 produce spermatozoa with defective pseudopodial projections, and these spermatozoa are largely immotile. Mutants in the spermatozoa do not translocate. Thus pseudopod movement is correlated with the presence of normal projections. Twelve mutants with defective muscles have spermatozoa with normal movement, so these genes do not specify products needed for both muscle and nonmuscle cell motility.

Biological responsiveness to the phorbol esters and specific binding of [3H]phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system.

Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. [3H]phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound.