Luehr, Sebastian et al. (2015) International Worm Meeting "Fluorescent CRISPR/Cas system to specifically label DNA sequences of the egl-1 locus."

Luehr, Sebastian, Conradt, Barbara

[International Worm Meeting, 2015]

Programmed cell death and in particular apoptosis is a crucial process in animal development and in the maintenance of cellular homeostasis. In Caenorhabditis elegans, the key activator of the central apoptotic pathway is the BH3-only protein EGL-1. EGL-1 is conserved and functions as a receiver of apoptotic stimuli [1-4]. EGL-1 activity is controlled at the level of expression; however, until now, the epigenetic regulation of egl-1 expression with regard to the chromatin state of its locus has not been analyzed yet.To address this, we plan to visualize specific DNA sequences of the egl-1 locus using the fluorescent CRISPR/Cas system to reveal their positions in the chromatin during embryonic development. To that end, we are using a system that has been developed for mammalian cells and adapt it for C. elegans by generating an inactive version of the Cas9 endonuclease fused with GFP (dCas9-GFP) [5,6]. As proof of concept, we are currently working on labeling the telomeres of developing embryos by co-expression of dCas9-GFP along with a small guide RNA (gRNA) targeting telomeric repeats.Generating multiple different gRNAs covering the egl-1 locus should provide sufficient labeling and can be applied to any loci lacking repetitive sequences. Due to the absence of cell deaths during the first embryonic cell divisions, we expect to see a condensed state of the egl-1 locus early during embryonic development, which presumably changes to an open state before the first wave of cell death (9th round of cell division). In case we can observe a change in chromatin state, we will investigate its role in the regulation of egl-1 expression.1. Conradt, B. & Horvitz, H. R. The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519-29 (1998).2. Nehme, R. & Conradt, B. egl-1: a key activator of apoptotic cell death in C. elegans. Oncogene 27 Suppl 1, S30-S40 (2008).3. Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59 (2008).4. Labi, V., Erlacher, M., Kiessling, S. & Villunger, a. BH3-only proteins in cell death initiation, malignant disease and anticancer therapy. Cell Death Differ. 13, 1325-1338 (2006).5. Anton, T., Bultmann, S., Leonhardt, H. & Markaki, Y. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system. Nucleus 5, 163-72 (2014).6. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479-1491 (2013).

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Yanjue Wu et al. (2005) International Worm Meeting "EGb 761 INHIBITS AMYLOID BETA OLIGOMERIZATION AND TOXICITY IN TRANSGENIC CAENORHABDITIS ELEGANS"

Christopher D Link, Yves Christen, Astrid G Zepeda, Yanjue Wu, Zhixin Wu, Yuan Luo

[International Worm Meeting, 2005]

Amyloid peptide aggregation has been postulated to link oxidative stress and neurodegeneration seen in Alzheimer's diesease (AD). We have previously demonstrated that the standardized Ginkgo Biloba extract, EGb 761, inhibits A aggregation in vitro and attenuates the expression of a stress response gene small heat shock protein (hsp 16-2) in Caenorhabditis elegans. In this study, we use EGb 761 as a pharmacological modulator to associate A species with the levels of oxidative free radicals and with A-induced toxicity in an A-expressing transgenic C.elegans model, which can express A in muscle cells after temperature up-shift. We observed that EGb 761 alleviated A-induced toxicity which correlated with its ability to specifically inhibit A oligomerization. Furthermore, EGb 761 reduces intracellular levels of hydrogen peroxide in the transgenic C.elegans. These findings suggest that A oligomers and A-induced oxidative strss are crucial for A toxicity, and EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.

Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Joost Monen, Arshad Desai, Francie Hyndman, Karen Oegema, Paul Maddox

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

Christian Rocheleau et al. (2006) Development & Evolution Meeting "Identification of components of an endogenous small-RNA-mediated pathway and chromatin remodeling proteins as enhancers of Ras signaling in C. elegans"

Meera Sundaram, Kevin Cullison, Christian Rocheleau, Yunling Wang, Yelena Bernstein

[Development & Evolution Meeting, 2006]

Endogenous small-RNA-mediated pathways and chromatin remodeling complexes have been shown to cooperate to negatively regulate gene expression. A screen for positive regulators of Ras signaling identified a number of small RNA regulatory proteins, including an Argonaute, a Dicer-related helicase, a RNA-directed RNA polymerase and a tudor domain protein which may constitute components of a novel small-RNA regulated pathway, as well as a number of chromatin remodeling proteins, including components of the NuA4 histone acetylase complex. Our current hypothesis is that these genes may function together in a common process to regulate the expression of a Ras pathway component in a tissue specific manner. We are testing candidate targets with hopes of identifying the small RNA(s) involved and to understand the mechanism of gene regulation.

Vayndorf, Elena et al. (2017) International Worm Meeting "C. elegans as a model for age-related phenotyping, compound screening and toxicological assessment."

Hincks, Joshua, Vayndorf, Elena, Taylor, Barbara, Harris, Michael

[International Worm Meeting, 2017]

The C. elegans pharynx is a tubular, bi-lobed, muscular pump encased in a basement membrane. The pharynx muscle contracts and relaxes as the animal feeds, generating a characteristic electrical signal that can be recorded as an "electropharyngeogram". The frequency of pharyngeal pumping decreases with age and is considered a reliable index of overall health. Recently, NemaMetrix has made available a device that easily monitors and quantifies the C. elegans electropharyngeogram. We have designed a two-week teaching module using the NemaMetrix platform in guided undergraduate research. Projects assay a locally relevant environmental toxin, a natural product, and the effect of age in C. elegans. We provide examples of research projects that can be done in a two-week research module as part of a biomedically-related undergraduate course.

Inoue, Takao (2011) International Worm Meeting "Possible regulation of bed-3 by blmp-1."

Inoue, Takao

[International Worm Meeting, 2011]

blmp-1 encodes the C. elegans ortholog of Blimp1, a SET-domain containing transcription factor. The modENCODE project identified a binding site for BLMP-1 in the intron 3 of the bed-3 gene, within a region where a vulva and hypodermis specific enhancer element was previously identified. We found that RNAi against blmp-1 causes down-regulation of a bed-3::gfp reporter containing intron 3. Moreover RNAi against blmp-1 caused a molting defect similar to a strong bed-3 mutant. These results suggest that blmp-1 regulates bed-3.

Jayamani, Elamparithi et al. (2015) International Worm Meeting "High-Throughput Screening (HTS) assay for the discovery of anti-infective agents against Acinetobacter baumannii infection."

Rajamuthiah, Rajmohan, Ausubel, Frederick M, Vilcinskas, Andreas, Fuchs, Beth Burgwyn, Mylonakis, Eleftherios, Jayamani, Elamparithi, Conery, Annie L., Larkins-Ford, Jonah

[International Worm Meeting, 2015]

Pan-drug resistant Acinetobacter baumannii is one of the most dangerous multidrug-resistant (MDR) microbes named as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found that A. baumannii is pathogenic and lethal to the model host organism Caenorhabditis elegans and have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective against A. baumannii. The screening assay involves coincubating C. elegans with A. baumannii in 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z' factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhance C. elegans survival in the presence of A. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquito Aedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 mug/ml) against A. baumannii. BR003-cecropin A causes membrane permeability in A. baumannii, which could be the underlying mechanism of its lethality. We have found that even at higher concentration (10X MIC), BR003-cecropin A is not toxic to the worms. Thus, BR003-cecropin A is potential AMP against A. baumannii infection.