A size threshold governs Caenorhabditis elegans developmental progression

The growth of organisms from humans to bacteria is affected by environmentalconditions. However, mechanisms governing growth and size control are not wellunderstood, particularly in the context of changes in food availability in developingmulticellular organisms. Here, we use a novel microfluidic platform to study theimpact of diet on the growth and development of the nematode Caenorhabditiselegans. This device allows us to observe individual worms throughoutlarval development, quantify their growth as well as pinpoint the moultingtransitions marking successive developmental stages. Under conditions of low foodavailability, worms grow very slowly, but do not moult until they have achieved athreshold size. The time spent in larval stages can be extended by over an order ofmagnitude, in agreement with a simple threshold size model. Thus, a critical wormsize appears to trigger developmental progression, and may contribute to prolongedlifespan under dietary restriction.     

Biochemical Analysis of Caenorhabditis elegans Surface Mutants

A collection of Caenorhabditis elegans mutants that show ectopic surface lectin binding (Srf mutants) was analyzed to determine the biochemical basis for this phenotype. This analysis involved selective removal or labeling of surface components, specific labeling of surface glycans, and fractionation of total protein with subsequent detection of wheat germ agglutinin (WGA) binding proteins. Wild-type and mutant nematodes showed no differences in their profiles of extractable surface glycoproteins or total WGA-binding proteins, suggesting that the ectopic lectin binding does not result from the novel expression of surface glycans. Instead, these results support a model in which ectopic lectin binding results from an unmasking of glycosylated components present in the insoluble cuticle matrix of wild-type animals. To explain the multiple internal defects found in some surface mutants, we propose that these mutants have a basic defect in protein processing. This defect would interfere with the expression of the postulated masking protein(s), as well as other proteins required for normal development.     

Measuring Movement to Determine Physiological Roles of Acetylcholinesterase Classes in Caenorhabditis elegans

A difference in movement has been hypothesized to exist between Caenorhabditis elegans strains lacking one of two main genes for acetylcholinesterase (AChE), ace-1(+) and ace-2(+). We explored the precision of movement as an endpoint by measuring and comparing the movements of these strains (VC505 and GG202, respectively) and of N2 (wild-type). The order of movement of the strains is: N2 > VC505 > GG202; therefore, loss of the ace-2(+) gene is more detrimental to movement. We then compared the sensitivities of the three strains to an AChE inhibitor (propoxur) by generating movement-concentration curves, identifying effective concentrations that decreased movement by 50% (EC₅₀), and comparing them. EC₅₀ show an order of: N2 ≈ GG202 < VC505. Therefore, the enzymes encoded by ace-1(+) were more susceptible to propoxur than those of ace-2(+), suggesting that the innate difference in the AChE classes'' contributions to movement will not always determine the strain sensitivity. Measuring movement was sufficiently precise to record differences following genetic manipulation and further chemical exposure.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.     

Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH₂-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Genome-wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development

The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-β signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.Subject Categories: Autophagy & Cell Death; Signal Transduction; Membrane & Intracellular Transport     

A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination

Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP.     

Localization of Cuticular Binding Sites of Concanavalin A on Caenorhabditis elegans and Meloidogyne incognita

Utilizing a Concanavalin A (Con A)-hemocyanin conjugate, the majority of cuticular Con A binding sites were shown to be localized on the head region of Caenorhabclitis elegans and Meloidogyne incognita. Secretions which apparently emanated from the amphids and inner labial papillae did not label.     

Toxicity of Glucosinolates and Their Enzymatic Decomposition Products to Caenorhabditis elegans

An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC₅₀) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC₅₀ value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC₅₀ value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

Synaptic polarity depends on phosphatidylinositol signaling regulated by myo-inositol monophosphatase in Caenorhabditis elegans

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP(2) on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP(2) synthesis through inhibition of IMPase. We propose that the PIP(2) signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity.     

Crossover Heterogeneity in the Absence of Hotspots in Caenorhabditis elegans

Crossovers play mechanical roles in meiotic chromosome segregation, generate genetic diversity by producing new allelic combinations, and facilitate evolution by decoupling linked alleles. In almost every species studied to date, crossover distributions are dramatically nonuniform, differing among sexes and across genomes, with spatial variation in crossover rates on scales from whole chromosomes to subkilobase hotspots. To understand the regulatory forces dictating these heterogeneous distributions a crucial first step is the fine-scale characterization of crossover distributions. Here we define the wild-type distribution of crossovers along a region of the C. elegans chromosome II at unprecedented resolution, using recombinant chromosomes of 243 hermaphrodites and 226 males. We find that well-characterized large-scale domains, with little fine-scale rate heterogeneity, dominate this region’s crossover landscape. Using the Gini coefficient as a summary statistic, we find that this region of the C. elegans genome has the least heterogeneous fine-scale crossover distribution yet observed among model organisms, and we show by simulation that the data are incompatible with a mammalian-type hotspot-rich landscape. The large-scale structural domains—the low-recombination center and the high-recombination arm—have a discrete boundary that we localize to a small region. This boundary coincides with the arm-center boundary defined both by nuclear-envelope attachment of DNA in somatic cells and GC content, consistent with proposals that these features of chromosome organization may be mechanical causes and evolutionary consequences of crossover recombination.     

Effects of Aldicarb and Fenamiphos on Acetycholinesterase and Motility of Caenorhabditis elegans

The ability of Caenorhabditis elegans to recover from exposure to high doses of aldicarb and fenamiphos was examined at the organismal and biochemical levels by determination of movement and acetylcholinesterase activity. Nematodes recovered rapidly from a 24-hour exposure to both compounds at concentrations that caused complete paralysis. Acetylcholinesterase regained nearly full activity after a 24-hour exposure to aldicarb but only 10% activity after exposure to fenamiphos. The nematodes were able to move normally, however, on the limited activity that was regained after fenamiphos treatment. Mutant C. elegans strains deficient in various molecular forms of acetylcholinesterase were utilized to demonstrate that the mechanism of recovery did not involve new synthesis of enzyme. This result was confirmed by experiments on acetylcholinesterase reactivation from live versus dead nematodes.     

Caenorhabditis elegans neuron degeneration and mitochondrial suppression caused by selected environmental chemicals

Mitochondrial alterations have been documented for many years in the brains of Parkinson’s disease (PD), a disorder that is characterized by the selective loss of dopamine neurons. Recent studies have demonstrated that Parkinson’s disease-associated proteins are either present in mitochondria or translocated into mitochondria in response to stress, further reinforcing the importance of the mitochondrial function in the pathogenesis of Parkinson’s disease. Exposure to environmental chemicals such as pesticides and heavy metals has been suggested as risk factors in the development of Parkinson’s disease. It has been reported that a number of environmental agents including tobacco smoke and perfluorinated compounds, pesticides, as well as metals (Mn²⁺ and Pb²⁺) modulate mitochondrial function. However the exact mechanism of mitochondrial alteration has not been defined in the context of the development and progression of Parkinson’s disease. The complexity of the mammalian system has made it difficult to dissect the molecular components involved in the pathogenesis of Parkinson’s disease. In the present study we used the nematode Caenorhabditis elegans (C. elegans) model of neuron degeneration and investigated the effect of environmental chemicals on mitochondrial biogenesis and mitochondrial gene regulation. Chronic exposure to low concentration (2 or 4 μM) of pesticide rotenone, resulted in significant loss of dopamine neuron in C. elegans, a classic feature of Parkinson’s disease. We then determined if the rotenone-induced neuron degeneration is accompanied by a change in mitochondria biogenesis. Analysis of mitochondrial genomic replication by quantitative PCR showed a dramatic decrease in mitochondrial DNA (mtDNA) copies of rotenone-treated C. elegans compared to control. This decreased mitochondrial biogenesis occurred prior to the development of loss of dopamine neurons, and was persistent. The inhibition of mtDNA replication was also found in C. elegans exposed to another neuron toxicant Mn²⁺ at the concentration 50 or 100 mM. We further examined the mitochondrial gene expression and found significant lower level of mitochondrial complex IV subunits COI and COII in C. elegans exposed to rotenone. These results demonstrate that environmental chemicals cause persistent suppression of mitochondrial biogenesis and mitochondrial gene expression, and suggest a critical role of modifying mitochondrial biogenesis in toxicants-induced neuron degeneration in C. elegans model.     

New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans

Although a growing number of studies have reported the importance of SUMOylation in genome maintenance and DNA double-strand break repair (DSBR), relevant target proteins and how this modification regulates their functions are yet to be clarified. Here, we analyzed SUMOylation of ZTF-8, the homolog of mammalian RHINO, to test the functional significance of this protein modification in the DSBR and DNA damage response (DDR) pathways in the Caenorhabditis elegans germline. We found that ZTF-8 is a direct target for SUMOylation in vivo and that its modification is required for DNA damage checkpoint induced apoptosis and DSBR. Non-SUMOylatable mutants of ZTF-8 mimic the phenotypes observed in ztf-8 null mutants, including reduced fertility, impaired DNA damage repair, and defective DNA damage checkpoint activation. However, while mutants for components acting in the SUMOylation pathway fail to properly localize ZTF-8, its localization is not altered in the ZTF-8 non-SUMOylatable mutants. Taken together, these data show that direct SUMOylation of ZTF-8 is required for its function in DSBR as well as DDR but not its localization. ZTF-8’s human ortholog is enriched in the germline, but its meiotic role as well as its post-translational modification has never been explored. Therefore, our discovery may assist in understanding the regulatory mechanism of this protein in DSBR and DDR in the germline.     

Fluoroacetic Acid Is a Potent and Specific Inhibitor of Reproduction in the Nematode Caenorhabditis elegans

Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent and specific inhibitor of reproduction in a bioassay using the nematode Caenorhabditis elegans. Fluoroacetic acid added to the growth medium reduced reproduction in the second generation by 50% at concentrations 3,000 times lower than the concentrations that reduced 24-hour survival by 50%. Four concentrations (2, 4, 8, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations, the survival rates were unaffected, and first-generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. Malonate, which inhibits the Krebs cycle, and itaconate, which inhibits the glyoxylate cycle, were tested individually and in combination. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid. Fluoroacetic acid shows promise as a tool in studies requiring age synchrony.