The location of the major protein in Caenorhabditis elegans sperm and spermatocytes

Using an affinity-purified antibody to the major sperm protein (MSP) in Caenorhabditis elegans sperm we have shown by immunofluorescence that the MSP is localized in the fibrous bodies of spermatocytes and early spermatids, in the cytoplasm of late spermatids, and in the pseudopods of spermatozoa. The MSP can also form crystalline inclusions in mutant and wild-type sperm. The function of this protein is still unknown, but its ability to form filaments and its localization in the pseudopod, together with the lack of actin in these sperm suggest that the MSP may be required for amoeboid motility.     

Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.     

Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans

In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. Although many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. Here we analyze microtubule behavior in Caenorhabditis elegans oocytes at early stages of the meiotic maturation process using confocal microscopy and live-cell imaging. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. We show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. We present evidence that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic G alpha(o/i) and G alpha(s) pathways and gap-junctional communication with somatic cells of the gonad. We propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD.     

First identification of a phosphorylcholine-substituted protein from Caenorhabditis elegans: isolation and characterization of the aspartyl protease ASP-6

Caenorhabditis elegans is a widely accepted model system for parasitic nematodes, drug screening and developmental studies. Similar to parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins carrying, in part, phosphorylcholine (PCho) substitutions, which might play important roles in nematode development, fertility and, at least in the case of parasites, survival within the host. With the exception of a major secretory/excretory product from Acanthocheilonema viteae (ES-62), no protein carrying this epitope has been studied in detail yet. Here we report on the identification, characterization and localization of the aspartyl protease ASP-6 of C. elegans, which is excreted by the nematode in a PCho-substituted form. Within the worm, most prominent expression of the protein is observed in the intestine, while muscle and epithelial cells express asp-6 to a lesser extent. In animals harboring an ASP-6::GFP fusion protein, diffuse fluorescence throughout the body cavity of adult worms indicates that the chimeric protein is secreted.     

Cuticle of Caenorhabditis elegans: its isolation and partial characterization

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross-linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.     

The astacin protein family in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.     

Direct isolation of longevity mutants in the nematode Caenorhabditis elegans

We have isolated several new EMS-induced, long-lived mutants of Caenorhabditis elegans, using a novel screen that eliminates the need for replica plating. Three new alleles of age-1 (z10, z12, and z25) were identified by failure to complement age-1 (hx546) for life span extension; these alleles had life spans ranging from 18.9 to 25.9 days at 25°C, with an average 46% increase in life span. After backcrossing, alleles were examined in a wild-type background for resistance to several environmental stresses: heat (35°C), ultraviolet (UV) light (20 J/m²), and hydrogen peroxide (H₂O₂) (0.5 M). Two replicates of the test of thermotolerance were completed on each strain, giving mean survivals of 842 min (hx546), 810 min (z10), 862 min (z12), and 860 min (z25), compared to 562 min for wild type. All the age-1 alleles were significantly tolerant, compared with wild type (P < 0.001). Two replicates for UV resistance were also completed with mean survivals of 103, 118, 108, and 89 hr, respectively, compared to 72 hr for wild type. One test of hydrogen peroxide resistance has shown that z12 and N2 had a mean survival of 41 hr, while the other age-1 alleles had mean survival of 54 hr (z10), and 62 hr (z25); H₂O₂ resistance is the only environmental stress that differentiates among the age-1 alleles. © 1996 Wiley-Liss, Inc.     

Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex

Microtubules are polymers of tubulin heterodimers that exhibit dynamic instability: periods of growth followed by periods of shrinkage. However, the molecular regulation of dynamic instability remains elusive. Here, we show that EFA-6, a cortically-localized protein, limits the growth of microtubules near the cell cortex of early embryonic cells from Caenorhabditis elegans, possibly by inducing microtubule catastrophes. Compared with wild type, embryos lacking EFA-6 had abnormally long and dense microtubules at the cell cortex, and growing microtubule plus ends resided at the cortex for up to five-fold longer. Loss of EFA-6 also caused excess centrosome separation and displacement towards the cell cortex early in mitosis, and subsequently a loss of anaphase spindle-pole oscillations and increased rates of spindle elongation. The centrosome separation phenotype was dependent on the motor protein dynein, suggesting a possible link between the modulation of microtubule dynamics at the cortex and dynein-dependent force production. EFA-6 orthologues activate ARF6-type GTPases to regulate vesicle trafficking. However, we show that only the C. elegans EFA-6 amino-terminus is both necessary and sufficient to limit microtubule growth along the cortex, and that this function is independent of ARF-6.     

Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans

The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer.     

Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.     

Cloning of a yolk protein gene family from Caenorhabditis elegans

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA-dependent. The rhsA locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhsA are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhsA or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed "transductional walking", by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.     

Prion protein in Caenorhabditis elegans: Distinct models of anti-BAX and neuropathology

The infectious agent of prion diseases is believed to be nucleic acid-free particles composed of misfolded conformational isomers of a host protein known as prion protein (PrP). Although this “protein-only” concept is generally accepted, decades of extensive research have not been able to elucidate the mechanisms by which PrP misfolding leads to neurodegeneration and infectivity. The challenges in studying prion diseases relate in part to the limitations of mammalian prion models, which include the long incubation period post-infection until symptoms develop, the high expense of maintaining mammals for extended periods, as well as safety issues. In order to develop prion models incorporating a genetically tractable simple system with a well-defined neuronal system, we generated transgenic C. elegans expressing the mouse PrP behind the pan-neuronal ric-19 promoter (P_ric-19). We show here that high expression of P_ric-19::PrP in C. elegans can result in altered morphology, defective mobility and shortened lifespan. Low expression of P_ric-19::PrP, however, does not cause any detectable harm. Using the dopamine neuron specific promoter P_dat-1, we also show that expression of the murine BAX, a pro-apoptotic member of the Bcl-2 family, causes dopamine neuron destruction in the nematode. However, co-expression of PrP inhibits BAX-mediated dopamine neuron degeneration, demonstrating for the first time that PrP has anti-BAX activity in living animals. Thus, these distinct PrP-transgenic C. elegans lines recapitulate a number of functional and neuropathological features of mammalian prion models and provide an opportunity for facile identification of genetic and environmental contributors to prion-associated pathology.Key words: prion, C. elegans, protein misfolding, cytoprotection, BAX, neurodegeneration     

A highly conserved nematode protein folding operon in Caenorhabditis elegans and Caenorhabditis briggsae

In the free-living model nematode, Caenorhabditis elegans, a protein-folding co-transcribed gene pair has previously been described. The degree and form of trans-splicing, orientation and spacing of the genes, and the co-ordinate co-expression of protein folding catalysts in the nematode's hypodermis indicated this to be a functionally important operon. This gene pair has now been cloned and compared in the related organism Caenorhabditis briggsae to identify evolutionarily conserved, functionally important features. The corresponding C. briggsae gene pair was found to share the operon-specific features, including sequence homology blocks in the upstream 5′ flanking regions. The intergenic regions were not conserved. The homology block closest to the translational initiation codon of the upstream gene was found to contain a known Ceanorhabbitis promoter element site, and may therefore be an important cis-regulatory region directing the hypodermis-specific expression of this operon gene of C. elegans. This study also provides further confirmation of the high degree of chromosomal synteny between these nematode species.     

5'-AMP-activated protein kinase signaling in Caenorhabditis elegans

5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.