115 Works

UNC-42 function is required for the ectopic expression of ASH markers in mls-2 mutant animals

Jordan F. Wood & Denise M. Ferkey
The HMX/NKS homeodomain transcription factor MLS-2 is required to initiate the expression of the AWC terminal selector ceh-36, and as such the AWCs of mls-2 loss-of-function mutant animals fail to express downstream AWC-specific terminal differentiation genes (Kim et al., 2010).  Interestingly, loss of mls-2 function was also shown to result in ectopic expression of ASH markers (sra-6p::gfp and osm-10p::gfp) in at least one neuron in a large percentage of mls-2 mutant animals (Kim et al., 2010).  The cells that ectopically express...

New alleles of C. elegans gene cls-2 (R107.6), called xc3, xc4, and xc5

Nicholas R. Munoz, Christopher J. Black, Ethan T. Young & Diana S. Chu
We have generated novel mutant alleles, named xc3, xc4, and xc5, of the gene cls-2 (R107.6) that encode one of the three predicted orthologs of mammalian CLASPs and of Drosophila ORBIT/MAST, microtuble-binding proteins (Akhmanova et al., 2001; Maiato et al., 2002). In C. elegans CLS-2 is required for meiosis and mitosis (Cheeseman et al., 2005; Dumont et al., 2010; Espiritu et al., 2012; Maton et al., 2015; Nahaboo et al., 2015). The alleles were isolated...

Rapamycin-induced protein dimerization as a tool for C. elegans research

Sriyash Mangal, Jeffrey Zielich, Eric J. Lambie & Esther Zanin
Induced protein dimerization is an invaluable tool in cellular biology to study protein function. Induced dimerization has been widely used to modulate enzymatic activity as well as expression and localization of proteins (Voß et al., 2015; DeRose et al., 2013). A popular method employs the chemical dimerizer rapamycin to induce binding between the FRB domain of the mTOR kinase and the FKBP12 protein (FK506 binding protein 12 kDa) (Putyrski and Schultz, 2012). Rapamycin-induced protein dimerization...

Loss of eif-2alpha phosphorylation on S49 (mammalian S51) associated with the integrated stress response hastens development in C. elegans​

Jarod Rollins, Noah Lind & Aric N. Rogers
The gene eif-2alpha (Y37E3.10) in C. elegans encodes for the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). The eIF2 trimer is required for delivering charged tRNAMet to the ribosome in a reaction requiring the hydrolysis of GTP to GDP (Sokabe et al., 2012). The phosphorylation of eif2-alpha on its 49th serine residue (Nukazuka et al., 2008) prevents replenishing GDP with GTP and thus reduces translation initiation by preventing delivery of tRNAMet. The phosphorylation...

SapTrap vectors for introducing point mutations with unc-119+ selection

Matthew L Schwartz & Erik M Jorgensen
SapTrap assembly generates CRISPR targeting vectors using a Golden Gate cloning strategy (Schwartz and Jorgensen, 2016). The original toolkit was designed for inserting tags, such as fluorescent proteins or affinity tags, into protein coding genes in C. elegans. In the original design, the floxed unc-119+ selectable marker was incorporated into the tag. For site-directed mutagenesis, the selectable marker is useful but a tag is unnecessary. We have developed a new set of vectors dedicated for...

Trehalose Extends Healthspan in C. elegans

Kristen J Robinson & Kathryn McCormick
Adult worms were reared and tested in M9 medium plus 5 mM trehalose, which has previously been shown to extend lifespan and increase pump frequency late in life in C. elegans (Honda et al., 2010). Using an alternate electrophysiological readout of pumping, electrophayrngeograms (EPGs), we examined pump frequency at earlier timepoints than previously reported. Pumps were stimulated with 10mM 5HT in M9, recorded as EPGs in a microfluidic device, and analyzed using NemAnalysis software (NemaMetrix)....

Erratum: aptf-1 mutants are primarily defective in head movement quiescence during C. elegans sleep

Bryan Robinson, Desiree L Goetting, Janine Cisneros Desir & Cheryl Van Buskirk
Robinson, B; Goetting, DL; Cisneros Desir, J; Van Buskirk, C (2019). aptf-1 mutants are primarily defective in head movement quiescence during C. elegans sleep. microPublication Biology. 10.17912/micropub.biology.000148 published online Aug 19, 2019 @ 09:06; corrected after publication Aug 22, 2019. In the version of the microPublication originally published online, the names of the third and last author were incorrect. The correct names are Janine Cisneros Desir and Cheryl Van Buskirk.  These mistakes have been corrected in the html and PDF versions of the...

Erratum: Endocannabinoid 2-arachidonoyl glycerol increases the transcription of daf-7 in ASI neurons

Celina Galles, Gastón M. Prez & Diego de Mendoza
Galles, C; Prez, GM; de Mendoza, D (2018). Endocannabinoid 2-arachidonoyl glycerol increases the transcription of daf-7 in ASI neurons. microPublication Biology. 10.17912/micropub.biology.000056 first published online Jul 29, 2018 @ 02:37; corrected after publication Aug 22, 2019. In the version of the microPublication originally published online, the name of the second author was misspelled. The correct spelling is Gastón M. Prez. This mistake has been corrected in the html and PDF versions of the microPublication.

Precision deletion of the entire coding sequence of the mod-5 locus causes increase in pharyngeal pumping frequency

Trisha Brock, Stelian Pop, Chandler Bradford, Jenn Lawson, Lauren Resch & Christopher Hopkins
​The C. elegans mod-5 gene encodes a serotonin transporter. The putative null allele contains a 1,688bp deletion leading to a premature stop codon in the resulting sequence (Ranganathan, 2001); however, unspliced the gene is 12,557bp in length from the start to the stop codon. We used CRISPR/Cas9 technology to create a 12,775bp deletion that eliminates all of the mod-5 coding sequence from the genome (Figure 1A). The mod-5(knu383) animals were measured for pharynx pumping frequency...

Silencing the ASI gustatory neuron pair increases expression of the stress-resistance gene sod-3 in a daf-16 and daf-3 independent manner

Peter Chisnell & Cynthia Kenyon
Ablation of the gustatory neuron pair ASI has been shown to extend lifespan, and this lifespan extension requires the function of the transcription factor daf-16/FOXO (Alcedo and Kenyon 2004). Given this relationship, we tested whether silencing the neuron pair ASI with the tetanus toxin light chain (Tetx), as opposed to ablating it, could increase the expression of a well- known DAF-16 target, the stress-resistance gene sod-3 (Furuyama et al. 2000; Honda and Honda 1999). Tetanus...

Basal pharyngeal pumping elevated in C. elegans mod-5 mutants

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In C. elegans, the reuptake of serotonin (5-HT) is facilitated by mod-5, which encodes a 5-HT transporter that is orthologous to a human 5-HT transporter (SLC6A4). mod-5 has been shown to effect both feeding and locomotion in C. elegans (Ranganathan et al., 2001; Jafarau et al. 2011). We obtained and analyzed EPG data using a microfluidic device (NemaMetrix) for mod-5 null mutant strains, mod-5(n3314) (A, Exp 1 n=15; Exp 2 n=32) and mod-5(n822), (B, Exp...

UNC-108/RAB-2 is required for C. elegans stress-induced sleep

Bryan Robinson & Cheryl Van Buskirk
In a genetic screen for mutants defective in stress-induced sleep (SIS) we isolated csn2, an allele of the UNC-108/RAB-2 GTPase. The point mutation in unc-108(csn2) is identical to that in the previously characterized loss-of-function allele unc-108(n3263), substituting a glutamine in place of a glycine that is conserved among Ras superfamily members (Mangahas et al., 2008). Similar to other unc-108(lf) mutants, csn2 animals move slowly (not shown). Here we show that unc-108(csn2) as well as previously...

An isoform-specific allele of the sax-7 locus

Dylan Rahe, Ines Carrera, Filip Cosmanescu & Oliver Hobert

Establishment of a CRISPR/Cas9-based strategy for inducible protein dimerization

Jeffrey Zielich, Sriyash Mangal, Esther Zanin & Eric J. Lambie
We modified Norris’s mCherry-tag repair donor vector by fusing a C. elegans codon-optimized fkbp12 sequence 3’ to mCherry (Mangal et al. 2018) and re-establishing the critical NotI site for 3’ homology region insertion (Figure 1 A; pEL226). This vector can be used to C-terminally tag any GOI by following the protocol of Norris et al. (2015), including subsequent Cre-mediated excision of the dual marker selection cassette (Figure 1 B). As proof of principle we C-terminally...

A quantitative trait locus for nictation behavior on chromosome V

Jun Kim, Daehan Lee & Junho Lee
Lee et al., 2017 showed that CB4856 has a lower nictation ratio (the number of nictating dauers over the number of moving dauers on a micro-dirt chip) than N2, and that a genetic locus (nict-1) on chromosome IV mediates this phenotype difference. Although this paper had no evidence of any other genetic loci for nictation behavior, we have studied other quantitative trait loci (QTL) using different linkage-based mapping strategies. Here, we report another genetic locus...

Localization of tbg-1 mRNAs and GFP::TBG-1 protein in Early C. elegans Embryos

Lauren DeMeyer & Mi Hye Song
tbg-1 encodes gamma-tubulin, a ubiquitous and highly conserved component of centrosomes in eukaryotic cells (Strome et al, 2001). Using smFISH we determined the localization of tbg-1 transcripts (red). tbg-1 transcripts are detected within distinct foci throughout the cytoplasm during both the first (A) and second (B) mitosis. tbg-1 transcripts are not enriched at centrosomes or either blastomere. In contrast, GFP tagged TBG-1 proteins (green signal; arrowheads) localize at centrosomes, as previously shown (Strome et al,...

me98 is a new allele of rad-54

Baptiste Roelens, Karl A Zawadzki & Anne M Villeneuve
We isolated the me98 mutant in a genetic screen for C. elegans mutants with an altered number of GFP::COSA-1 foci, which mark the sites of crossovers in wild-type C. elegans germ cells (ROSU et al. 2013). After multiple rounds of outcrossing, we confirmed that the me98 mutant is defective in meiotic prophase as i) chromosomes in diakinesis oocytes appear partially decondensed and structurally compromised (Fig. 1A), ii) me98 fails to form the six GFP::COSA-1 foci...

me101 is a new allele of rad-51

Baptiste Roelens, Karl A Zawadzki & Anne M Villeneuve
The me101 allele was isolated in a genetic screen for mutants with an altered number of GFP::COSA-1 foci, which mark the sites of crossovers in C. elegans germ cells (ROSU et al. 2013). After multiple rounds of outcrossing, we confirmed that me101 mutants were defective in some aspects of meiotic prophase, as late pachytene me101 mutant meiocytes failed to form the six GFP::COSA-1 foci observed in wild-type late pachytene meiocytes (Fig 1.A). We also observed...

sy680 is a novel allele of pkd-2

Allyson Whittaker, Gary Schindelman, Shahla Gharib & Paul W. Sternberg
A new allele of pkd-2 was isolated in a behavioral genetic screen for male mating defects, and found to result in a substitution of Arginine for Glycine in the equivalent of human PKD2 alanine 615. The C. elegans ortholog of polcystin-2 is encoded by pkd-2 (Barr et al., 2001). From an EMS screen of a plg-1; him-5 strain for male mating defective mutants and a secondary behavioral screen for defects in discrete steps of male...

oxi-1 and fshr-1 are required for neuromuscular signaling under normal and oxidative stress conditions in C. elegans

Barry Wei & Jennifer R Kowalski
Reactive oxygen species (ROS) contribute to neuronal degeneration by readily reacting with cellular components, consequently breaking down cellular integrity. Excess ROS often leads to oxidative stress, which results from destabilization of the organism's ability to control the balance between antioxidants and free radicals (Chandra et al. 2015). The ubiquitin-proteasome system helps to regulate oxidative stress and overall damage to cellular components by forming chains of ubiquitin polypeptides on cellular proteins; these chains then serve as...

Overexpression of hindsight in sensory organ precursors is associated with a transformation of campaniform sensilla to microchaetae in the Drosophila wing

Krzysztof Szablewski & Bruce H. Reed
The adult Drosophila wing blade contains sense organs known as campaniform sensilla. These cellular structures sense pressure or strain in the wing during flight and provide neural feedback required for coordinated wing movements (Bartussek and Lehmann, 2016). The dorsal surface of the wing blade includes three campaniform sensilla along wing vein L3, two on the first longitudinal vein near the tip of the costa, and a single sensillum on the anterior cross vein. Other campaniform...

Microfluidic EPG Recordings Show Striking Pharyngeal Pumping Phenotype in a C. elegans Alzheimer's Disease Model

Janis C Weeks, Kristen J Robinson, Benedicta Wanjeri, Philip F Copenhaver & William M Roberts
Strain CL4176 accumulates human amyloid-β1-42 in body wall muscles after animals are shifted from 15 to 25 oC, causing paralysis (Link et al. 2003). We temperature-shifted worms at early L4 and quantified pharyngeal pumping at 48 h using a microfluidic electropharyngeogram (EPG) recording platform (Lockery et al. 2012; Weeks et al. 2016). Recordings were made in M9 buffer with 10 mM serotonin and analyzed by automated pump-recognition software. At 48 h, control CL802 worms showed...

Asymmetric distribution of cyb-3 in 4-cell stage embryos.

W. Matthew Michael
Early embryos were fixed and stained with Mab F2F4 (green), shown to recognize CYB-3 (Michael, 2016), and DAPI, to illuminate the DNA (blue). Either wild type or par-4 mutant embryos were examined, after 24-hour incubation at 25C (the non-permissive temperature for the it47 allele of par-4). Anterior is to the left in all images. The data presented here reveals previously not shown data that depicts CYB-3 as asymmetrically distributed at the 4-cell stage. These data...

A C. elegans MAP kinase pathway is required for wild-type display of an L1-specific surface antigen (srf-6 is nsy-1 III)

Stephen J. Foley, Zheyang Wu & Samuel M. Politz
In previous work, we showed that three different srf-6 mutants carry mutations in gene nsy-1 (Van Sciver et al., 2019), and that a srf-6 mutant shows the 2AWCON phenotype characteristic of nsy-1 mutants. We also showed that srf-6(yj13) fails to complement nsy-1(ok593) (Honzel et al., 2019). We concluded that srf-6 and nsy-1 are the same gene. C. elegans gene nsy-1 encodes a MAP kinase kinase kinase (MAPKKK) that carries out the first step in a...

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