Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.     

Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes

Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments.     

Receptors and Other Signaling Proteins Required for Serotonin Control of Locomotion in Caenorhabditis elegans

A better understanding of the molecular mechanisms of signaling by the neurotransmitter serotonin is required to assess the hypothesis that defects in serotonin signaling underlie depression in humans. Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion, providing a genetic system to analyze serotonin signaling. From large-scale genetic screens we identified 36 mutants of C. elegans in which serotonin fails to have its normal effect of slowing locomotion, and we molecularly identified eight genes affected by 19 of the mutations. Two of the genes encode the serotonin-gated ion channel MOD-1 and the G-protein-coupled serotonin receptor SER-4. mod-1 is expressed in the neurons and muscles that directly control locomotion, while ser-4 is expressed in an almost entirely non-overlapping set of sensory and interneurons. The cells expressing the two receptors are largely not direct postsynaptic targets of serotonergic neurons. We analyzed animals lacking or overexpressing the receptors in various combinations using several assays for serotonin response. We found that the two receptors act in parallel to affect locomotion. Our results show that serotonin functions as an extrasynaptic signal that independently activates multiple receptors at a distance from its release sites and identify at least six additional proteins that appear to act with serotonin receptors to mediate serotonin response.     

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.     

Characterization of the chondroitin sulfates in wild type Caenorhabditis elegans

The purpose of this study was to isolate and characterize the GAGs from the wild type nematode Caenorhabditis elegans in preparation for the characterization of the transgenic form constructed by Link [Proc. Natl. Acad. Sci. USA 92 (1995) 9368] which expresses various forms of beta-peptide (or A4 peptide). This peptide forms deposits very similar to the ones found in the neuritic plaques and neurofibrillary tangles in Alzheimer disease (AD). Characterization has been accomplished by degradation with specific enzymes and analysis of the products by TLC and HPLC. The results were compared with earlier works and shown to differ in disaccharide content.     

Linking Gene Expression in the Intestine to Production of Gametes Through the Phosphate Transporter PITR-1 in Caenorhabditis elegans

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane-bound transporters and coupled to Na⁺ transport. Mammalian sodium-dependent Pi cotransporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than two decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the Caenorhabditis elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.     

Expression analysis of ABC transporters reveals differential functions of tandemly duplicated genes in Caenorhabditis elegans

We have previously identified 60 predicted ABC transporter genes in the Caenorhabditis elegans genome and classified them into eight groups. As an initial step towards understanding how these putative ABC genes work in worms, we generated promoter-fluorescent protein fusions for the entire family to address when and where these genes are turned on in vivo. Both Aequoria green fluorescent protein (GFP) and Discosoma red fluorescent protein (RFP) were used as reporters in our transgenic assay. Observable expression is more frequently seen from fusions to genes in subfamilies B, C, D and E than those in subfamilies A and G. Sixteen worm ABC genes are found in tandem duplications, forming two four-gene clusters and four two-gene clusters. Fifteen out of the 16 duplicated gene promoters drove different or partially overlapping expression patterns, suggesting active functions for these duplicated genes. Furthermore, our results suggest that an internal promoter can cause differential expression of genes within an operon. Finally, our observations suggest that it is possible for coding sequences to function as a regulatory region for a neighbouring gene.     

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.     

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.     

Characterization of a conserved apoptotic marker expressed in Caenorhabditis elegans phagocytic cells

We have identified and characterized a monoclonal antibody, F2-P3E3, that recognizes a Caenorhabditis elegans apoptotic epitope expressed within phagocytic cells, which is conserved in four other nematode species. In C. elegans, F2-P3E3 staining requires both programmed cell death and phagocytosis. We show that the F2-P3E3 epitope is expressed within embryonic intestinal cells, which act as phagocytes but do not undergo programmed cell death. F2-P3E3 staining is present within LMP-1::GFP labeled organelles in the intestinal primordium and is coincident with persistent DNA that has been phagocytosed in nuc-1(-) embryos, suggesting that it labels phagosomes. While apoptotic events are typically isolated in C. elegans, F2-P3E3 staining is commonly found within adjacent cells. This observation suggests that F2-P3E3 might recognize an epitope expressed in multiple cells in response to signals from a single corpse. F2-P3E3 represents a new tool for studying cell death in C. elegans.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Location of 2(')-O-methyl nucleotides in 26S rRNA and methylation guide snoRNAs in Caenorhabditis elegans

Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O-methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O-methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues.     

Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans

Widespread resistance to chemotherapeutic agents is one of the biggest challenges facing human health and the agricultural industry, with resistance to all current anthelmintics now recorded and few new agents or vaccines available. Understanding the development of drug resistance in parasitic nematodes is critical to prolonging the efficacy of current anthelmintics, developing markers for monitoring drug resistance and is beneficial in the design of new chemotherapeutic agents or targets. This study describes the development of ivermectin-resistant strains of the model nematode Caenorhabditis elegans through step-wise exposure to increasing doses of ivermectin commencing with a non-toxic dose of 1 ng/ml. Resistant strains were developed that displayed a multidrug resistance phenotype with cross-resistance to the related drug moxidectin and to other anthelmintics, levamisole and pyrantel, but not albendazole. Resistance was associated with increased expression of the multidrug resistance proteins (MRPs) and P-glycoproteins. Resistance to ivermectin was reversible by the co-administration of MRP, P-glycoprotein and glutathione biosynthesis inhibitors, confirming the involvement of these proteins in resistance. In our model, resistance to low levels of ivermectin (相似文献   

Antinematodal activity and the mechanism of the antimicrobial peptide, HP (2-20), against Caenorhabditis elegans

The peptide HP (2-20), derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1), has a nematicidal activity against eggs and worms of Caenorhabditis elegans. Eggs treated with HP (2-20) (69%) has a higher fluorescence intensity with propidium iodide staining, which was similar to that of melittin (82%) but higher than untreated cells (5.7%). Confocal microscopy showed that the peptides were located in the shell of the eggs and the inner and outer surfaces of the worms. HP (2-20) therefore may exert its antinematodal activity by disrupting the structure of the egg's shell and the cell membrane via pore formation or by direct interaction with the lipid bilayers in a detergent-like manner.     

Overexpression of PPK-1, the Caenorhabditis elegans Type I PIP kinase, inhibits growth cone collapse in the developing nervous system and causes axonal degeneration in adults

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP₂) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP₂ is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP₂ synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP₂in vitro and that overexpression of the kinase causes an increase in PIP₂ levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP₂ levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.