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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...

Mutations in KCNQ potassium channels cause pharyngeal pumping defects in C. elegans

Yoanne Clovis, Alexis Webb, Carl Turner & Bill Roberts
Pumps were stimulated with 10mM 5HT in M9 recorded as electropharyngeograms (EPGs) for 2 minutes in a NemaMetrix ScreenChip, and analyzed using NemAnalysis software (NemaMetrix). The null mutant strains kqt-1(aw3) and kqt-3 (aw1) were kindly donated by Dr. Aguan Wei (Wei et al., 2002) A) Pump frequency in kqt-1(aw3) animals was significantly lower than in N2s, while kqt-3(aw1) worms showed an increase in pump frequency (*p<0.05; ***p<0.01; 1-tailed Mann-Whitney U-test; n = 21-23 worms in...

Gene bashing of ceh-6 locus identifies genomic regions important for ceh-6 rectal cell expression and rescue of its mutant lethality

Arnaud Ahier, Shashi Kumar Suman & Sophie Jarriault
Loss of the activity of certain genes, such as ceh-6, can lead to lethality at early developmental stages, precluding the study of their function later on during development. Indeed, it was reported that more than 80% of ceh-6(mg60) animals died during embryogenesis exhibiting various phenotypes, including an abnormal rectal area and absent excretory canal cell (Bürglin and Ruvkun 2001). mg60 is a 1.4kb deletion allele that removes ceh-6 second exon and is believed to cause...

Variation between nematodes in a multi-sensory behavioral assay

Renae Ellis & Gareth Harris
An environment is often represented by numerous sensory cues. In order to better survive, an animal often needs to detect and process sensory cues simultaneously to make an appropriate behavioral decision. The Caenorhabditis elegans (C. elegans) genome encodes homologues of a significant number of molecules expressed in mammalian brains, which allows characterizing of the molecular and circuit basis for multi-sensory behavior during decision-making (Bargmann, 1998). In addition, studies have demonstrated various genes and neurons in...

Expression of an endosome-excluded Cd63 prevents axis elongation in Xenopus

Jennifer Kreis, Ramona Bonß, Kerstin Feistel & Philipp Vick
Cd63 is an adapter protein belonging to the tetraspanin family. As other members, it possesses four membrane-spanning domains, two extracellular loops, and the N- and C-terminus both face the cytoplasm. Cd63 is highly enriched in intraluminal vesicles (ILV) of late endosomes (LE; also called multivesicular bodies, MVB; Termini and Gillette, 2017). Cd63 trafficking to LE occurs via classical routes, i.e. from the ER to the Golgi complex, then to the plasma membrane, from where it...

The tetraspanin Cd63 is required for eye morphogenesis in Xenopus

Jennifer Kreis, Ramona Bonß & Philipp Vick
Tetraspanins (TSPAN) are a family of (33 in humans) scaffold proteins consisting of the name-giving four membrane-spanning domains, two extracellular loops, and an intracellular N- and C-terminus. By forming ‘tetraspanin-enriched microdomains’ they participate in organizing the plasma membrane and serve as multipurpose adapters. Some can be found ubiquitously in most cells, while others are specific to certain cell types. On a subcellular level, enrichment in distinct membrane-associated organelles has been documented for several members (Termini...

Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells

Anand K Singh, Jie Zhang, Daniel Hebenstreit & Saverio Brogna
UPF1 is an RNA helicase that scans RNA to unwind secondary structures and to displace associated factors (Franks et al., 2010; Fiorini et al., 2015; Lee et al., 2015; Kanaan et al., 2018). UPF1 has been mostly studied for its role in nonsense mediated mRNA decay (NMD) and other translation-dependent RNA quality-control pathways in the cytoplasm (Isken and Maquat, 2008; Kim and Maquat, 2019). However, we have recently reported that UPF1 associates, genome wide, with...

Single-cell resolution long-term luciferase imaging in cultivated Drosophila brains

Frank K. Schubert, Charlotte Helfrich-Förster & Dirk Rieger
In Drosophila melanogaster, approximately 150 so-called clock neurons express the molecular components of the circadian clock mechanism. The activity of these neurons enables the fly to anticipate and track cyclic changes in their environment and to respond to them accordingly. The core of the circadian clock is built by two interlocked transcriptional feedback loops. In the first loop, the transcription factors CLOCK (CLK) and CYCLE (CYC) initiate the expression of the core clock proteins PERIOD...

The salt-inducible kinase KIN-29 regulates lifespan via the class II histone-deacetylase HDA-4

Tatiana Nikooei, Aja McDonagh & Alexander M. van der Linden
kin-29 encodes the C. elegans homolog of mammalian Salt-Inducible Kinases (SIKs). kin-29 mutants are small, have increased propensity to develop into non-reproductive dauer larvae, have reduced chemoreceptor gene expression (Lanjuin and Sengupta, 2002; van der Linden et al.., 2007), have reduced cellular ATP despite increased fat stores, and show reduced sleep (Grubbs et al., 2020). KIN-29 phosphorylates and inhibits the class II histone deacetylase 4 homolog HDA-4 to regulate gene expression in sensory neurons (van...

Spontaneous neoplasia in the western clawed frog Xenopus tropicalis

Makoto Suzuki, Takeshi Igawa, Nanoka Suzuki, Hajime Ogino & Haruki Ochi
Xenopus tropicalis is an excellent model organism for studies on vertebrate development and regeneration (Horb et al., 2019) and is also useful for the study of tumor formation (Van Nieuwenhuysen et al., 2015; Naert et al., 2016). Spontaneously occurring neoplasia in amphibians have been reported, such as in X. laevis, Rana pipiens, and Andria japonicus (McKinnell et al., 1968; Meyer-Rochow et al., 1991; Kawasumi et al., 2012). In X. laevis, one of 4,000 frogs (a...

Loss of nucleosome remodelers CHRAC/ACF does not sensitize early Drosophila embryos to X-rays

Alessandro Scacchetti & Peter B. Becker
The ‘Chromatin Accessibility Complex’ (CHRAC) and ‘ATP-utilizing chromatin assembly and remodeling factor’ (ACF) of Drosophila melanogaster are chromatin remodeling complexes that slide nucleosomes (Becker and Horz, 2002). Both originate from the association of the ATPase ISWI and a large subunit ACF1. CHRAC contains two additional histone-fold subunits, CHRAC-14 and CHRAC-16 (Corona et al., 2000), but its nucleosome sliding activity is essentially similar to ACF in vitro (Hartlepp et al., 2005). CHRAC/ACF are implicated in several...

The C. elegans TRPV channel proteins OSM-9 and OCR-2 contribute to aversive chemical sensitivity

Emily A. Mehle, Savannah E. Sojka, Medha K.C., Rosy M. Zel, Sebastian J. Reese & Denise M. Ferkey
Transient receptor potential (TRP) channels are a family of cation channels that are important for response to diverse external stimuli across eukaryotes (Venkatachalam and Montell 2007; Samanta et al. 2018). In C. elegans, only members of the TRPV family, which includes osm-9, ocr-1, ocr-2, ocr-3 and ocr-4 (Colbert and Bargmann 1995; Colbert et al. 1997; Tobin et al. 2002), have been shown to play a role in chemosensory behavior (Bargmann 2006). OSM-9 and OCR-2 are...

Two new functions in the WormBase Enrichment Suite

David Angeles-Albores, Raymond Y.N. Lee, Juancarlos Chan & Paul W. Sternberg
​Genome-wide experiments routinely generate large amounts of data that can be hard to interpret biologically. A common approach to interpreting these results is to employ enrichment analyses of controlled languages, known as ontologies, that describe various biological parameters such as gene molecular or biological function. In C. elegans, three distinct ontologies, the Gene Ontology (GO), Anatomy Ontology (AO), and the Worm Phenotype Ontology (WPO) are used to annotate gene function, expression and phenotype, respectively (Ashburner...

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...

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...

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...

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...

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 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...

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...

The bodies of dpy-10(e128) are twice as stiff as wild type

Sylvia Fechner, Frédéric Loizeau, Adam L Nekimken, Beth L Pruitt & Miriam B Goodman
DPY-10 is a collagen protein in the nematode's cuticle. Mutations in the dpy-10 gene induce various morphological changes that lead to animals with a dumpy or roller phenotype (Levy, Yang & Kramer, 1993). Here, we asked how such mutations affect body stiffness by comparing force-indentation curves in dpy-10(e128) and wild type worms. On average, dpy-10(e128) worms have a steeper force-indentation relationship, leading to a higher body stiffness (Fig 1.C). Average stiffness values were 2.5 0.9...

Effect of the protease plasmin on C. elegans hyperactive DEG/ENaC channels MEC-4(d) and UNC-8(d)

Christina K. Johnson, David D. Miller & Laura Bianchi
MEC-4 and UNC-8 are subunits of the DEG/ENaC family of voltage-independent Na+ channels in C. elegans (Driscoll and Chalfie 1991, Canessa, Horisberger et al. 1993, Waldmann, Champigny et al. 1996, Waldmann, Champigny et al. 1997, de Weille, Bassilana et al. 1998, Waldmann and Lazdunski 1998). While MEC-4 is expressed in body touch neurons where it mediates the transduction of gentle touch sensation (Driscoll and Chalfie 1991, O'Hagan, Chalfie et al. 2005), UNC-8 is primarily expressed...

Deletion of a putative HDA-1 binding site in the hlh-2 promoter eliminates expression in C. elegans dorsal uterine cells

Taylor N Medwig-Kinney, Nicholas J Palmisano & David Q Matus
During C. elegans development, two cells, Z1 and Z4, give rise to the entire somatic gonad, including populations of dorsal uterine (DU) and ventral uterine (VU) cells, as well as the postmitotic anchor cell (AC), which invades the underlying basement membrane during uterine-vulval morphogenesis (Kimble & Hirsh, 1979; Sherwood & Sternberg, 2003). The helix-loop-helix transcription factor hlh-2 (E/Daughterless) is expressed in these three cell types, among others, and is particularly enriched in the AC following...

A novel null allele of C. elegans gene ceh-14

Emily A. Bayer & Oliver Hobert
We have generated a novel null allele, ot900, of the C. elegans LIM homeodomain gene ceh-14. All existing deletion alleles of ceh-14 were generated using a Tc1 transposon insertion, one of which, ch3, is a putative null based on the resulting frame-shift and a reduction in detectable mRNA and protein levels (Cassata et al. 2000). However, in our hands we have noticed a propensity of the ch3 allele to revert to wild-type after several generations,...

Expression pattern of endogenous PAR-4A & C after CRISPR/Cas9-mediated genome editing

Vincent Roy, Olivier Gagné, Karim Hamiche, Jean-Claude Labbé & Patrick Narbonne
PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al....

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  • Kurume University Medical Center
  • University of California, Santa Barbara
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  • Worcester Polytechnic Institute