28 Works

nu444 is a novel allele of pkc-1 in C. elegans

Han Wang & Derek Sieburth
Here, we report nu444 as a novel allele of the gene pkc-1 that encodes the protein kinase C-1 in C. elegans. The nu444 allele was originally isolated from a forward genetic screen for mutants that suppressed the “Hic” (Hypersensitivity to Inhibitors of Cholinesterase) phenotype of dgk-1(nu62) mutants, which had increased acetylcholine release at the neuromuscular junction (Sieburth et al., 2007). In this screen, several genes that are important for neuropeptide secretion were recovered, including pkc-1(nu448)...

daf-2 modulates regeneration of mechanosensory neurons I

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
Caenorhabditis elegans (C. elegans) possess the ability to spontaneously regenerate injured axons via a highly efficient mechanism known as axonal fusion (Ghosh-Roy, et al., 2010; Neumann et al., 2011; Neumann et al., 2015; Abay et al., 2017). Following laser axotomy, regrowth from the proximal axon segment (still attached to the cell body) reconnects and fuses with its separated distal segment (Fig. 1A). We recently demonstrated that the level of axonal fusion increases with age (Abay...

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...

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,...

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...

Screening for C. elegans male copulation-defective mutants by the mating plug phenotype

Katharine Liu, Yvonne Hajdu-Cronin, Ann Chen, Gary Schindelman, Allyson Whittaker, Shahla Gharib & Paul W. Sternberg
We describe an efficient screen for male mating defective mutants in Caenorhabditis elegans. We report the isolation of 20 alleles that confer specific mating defects. In a previously reported screen (Hajdu-Cronin et al, 2017), we isolated 19 Cod (for copulation defective) strains in which morphologically wild-type males fail to mate. Failure to mate could conceivably result from defects in any step of the mating process: response, turning, vulva location, spicule insertion, and sperm transfer. By...

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...

daf-2 regeneration of mechanosensory neurons: integration

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
We previously established that a critical event for axonal fusion to occur is the exposure of PS ‘save-me’ signals after injury (Neumann et al., 2015), which increase with advancing age (Abay et al., 2017a). To determine if daf-2 is involved in this age-dependent modulation of PS exposure, we visualised and quantified the level of PS exposed after PLM axotomy using a secreted, tagged version of Annexin V (Neumann et al., 2015; Mapes et al., 2012)....

xbp-1 mRNA splicing is attenuated under prolonged exposure to ER stress

Jennifer Tsialikas & Yair Argon
IRE-1 is an endoplasmic reticulum (ER) membrane-bound protein that mediates the unfolded protein response (UPR) when cells are under ER stress. Once activated, IRE-1 cleaves an intron from the xbp-1 mRNA (Calfon et al. 2002). This yields an active XBP-1 transcription factor, which activates transcription of ER chaperones and other genes involved in protein biosynthesis. Splicing of the xbp-1 intron can be measured by a shift in size in an agarose gel (Shen et al....

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...

Novel deletion alleles of a C. elegans gene Y73E7A.1, named as tm6429 and tm6475

Yuji Suehiro, Sawako Yoshina, Sayaka Hori & Shohei Mitani
We report tm6429 and tm6475 as novel deletion alleles of the gene Y73E7A.1 that is a homologue of mammalian Coiled-coil domain containing 124 (Ccdc124)1. The Ccdc124 is a conserved gene from invertebrates to human. In human cell lines, Ccdc124 is a component of the centrosome during interphase and at the G2/M transition. During cell division, Ccdc124 relocates to the midbody at telophase and acts as an essential molecular component in cytokinesis2. The alleles were isolated...

acdh-1 transcripts are expressed in the intestine

Dustin Updike
​Stellaris smFISH probes targeting acdh-1, a short-chain acyl-CoA dehydrogenase, are shown in red (Cal Fluor 610). DAPI/blue marks embryonic nuclei, and PGL-1::GFP shows the corresponding location of P granules surrounding germ cell nuclei (arrowheads, green). During embryogenesis (A), acdh-1 expression begins in the E cells (arrow, red). Expression continues in the developing intestine, shown in red, throughout embryogenesis. Intestinal expression of acdh-1 persists through larval development in the L1 (B) and L2 (C) stages. These...

Mapping results for a set of cGAL effectors and drivers

​Sophie J Walton, Han Wang, Jonathan Lui & Paul W Sternberg
Recently, the GAL4-UAS system (cGAL) has been adapted for use in C. elegans for control of gene expression across 15°C - 25°C (Wang et al., 2017). In order to create a desired gene expression pattern, one crosses a transgenic strain containing a driver construct with another strain containing an effector gene. Here we mapped several cGAL driver and effector integrations. We first crossed each of the cGAL driver and effector strains with N2 males, picked...

Copulation defective mutants of C. elegans

Yvonne M. Hajdu-Cronin, Katharine S. Liu, Leslie Barber, Helen M. Chamberlin, William Boorstein & Paul W. Sternberg
​Description To identify genes involved in male copulatory behavior, we carried out an F2 clonal screen in a him-5 mutant background. We identified 20 mutations that affect male mating behavior without causing gross defects in morphology. ​ Male mating in C. elegans comprises at least five steps (Liu and Sternberg, 1995). (l) The male responds to the hermaphrodite by backing his tail along the length of the hermaphrodite, (2) he turns over or under her...

Reduced pharyngeal pumping rates observed in tph-1 mutants using microfluidic electropharyngeogram (EPG) recordings

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In Caenorhabditis elegans, serotonin (5-HT) activates and controls pharyngeal pumping in response to food (Horvitz et al., 1982; Sze et al., 2000; Song and Avery 2012). Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping (Sze et al., 2000; Avery and Horvitz 1990; Song and Avery 2012). We used a microfluidic electropharyngeogram...

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...

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...

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...

Novel deletion alleles of a C. elegans gene Y48E1C.1, named as tm5468, tm5625 and tm5626

S; Suehiro Hori
We report tm5468, tm5625 and tm5626 as novel deletion alleles of the gene Y48E1C.1 that is the only ortholog of human calmodulin-lysine N-methyltransferase (CAMKMT)1. CAMKMT encodes an evolutionarily conserved enzyme class I protein methyltransferase that acts in the formation of trimethyllysine in calmodulin for calcium-dependent signaling2. CAMKMT mutation is associated with Hypotonia-cystinuria syndrome in human2,3. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV4. In the screening, all the alleles...

Extending the CRISPR toolbox for C. elegans: Cpf1 as an alternative gene editing system for AT-rich sequences

Annabel Ebbing, Peng Shang, Niels Geijsen & Hendrik C. Korswagen
The CRISPR/Cas9 system has become a powerful tool for genome-editing in C. elegans. Sequence specificity of the system is determined by a guide RNA which targets the Cas9 endonuclease to the genomic region of interest. In addition, Cas9 needs to interact with a so-called protospacer-adjacent motif (PAM) next to the target sequence. In case of Cas9 from Streptococcus pyogenes (SpCas9), the most commonly used Cas9, this sequence is NGG. Because the C. elegans genome sequence...

SPAS-1 expression in neurons and vulva during L4 stage

George Brown & Rachid El Bejjani
spas-1 is the worm homolog of the human spastic paraplegia gene, spastin. Previous work in C. elegans has shown that SPAS-1 disassembles microtubules in an ATP-dependent manner (Matsushita-Ishiodori et al, 2007). Another study shows that SPAS-1 plays a role in synapse remodeling in motor neurons (Kurup et al, 2015). Here we describe the expression of SPAS-1 in L4 hermaphrodite larvae using the transcriptional reporter, axrEx8 [Pspas-1:mCh]: A) Low magnification (10X objective) image of an entire...

Novel deletion alleles of a C. elegans gene C38D4.9, named as tm4476 and tm4561

Sawako Yoshina, Sayaka Hori, Yuji Suehiro & Shohei Mitani
We report tm4476 and tm4561 as novel alleles of C38D4.9 gene that is an ortholog of human METTL5 (methyltransferase like 5)1. Human METTL5 function has not been reported. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV2. In the screening, both the alleles were detected by nested PCR using the following primer sets, 5’-CCGCCTATATCATGGCGCTT-3’ and 5’-GCTGGTAGTTGCCACCGCAT-3’ for first round PCR and 5’-ACTATCGAAGCCGCGCTTCA-3’ and 5’-AACCCCAACTCCGTCCCATT-3’ for second round PCR. By Sanger...

A new mutation with a polycystin phenotypic spectrum in Caenorhabditis elegans

Allyson Whittaker, Gary Schindelman, Shahla Gharib & Paul W. Sternberg
lov-1 and pkd-2, which encode the C. elegans orthologs of human polycystin-1 and -2, are necessary for three particular aspects of male mating behavior. In a screen for male mating defective mutants with similar spectrum of mating defects, we identified a mutation that apparently defines a new locus, lov-3. We isolated the sy682 mutation in an ethyl-methane sulphonate (EMS)-screen of Caenorhabditis elegans strain PS1395 [genotype: plg-1(e2001d); him-5(e1490)] for mutant males that do not mate efficiently...

daf-2 modulates regeneration of mechanosensory neurons II

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
The daf-2 gene encodes an insulin-like growth factor/IGF-1 receptor that regulates C. elegans embryonic and larval development. It has previously been shown that DAF-2 inhibits neurite regeneration of the GABAergic motor neurons and PVD sensory neurons in an age-dependent fashion (Bryne et al., 2014; Kravtsov et al., 2017). Following injury, the posterior lateral microtubule (PLM) neurons are capable of regenerating through axonal fusion, a highly efficient regrowth mechanism in which separated fragments fuse back together...

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