Labouesse M et al. (2000) European Worm Meeting "che-14 encodes a sterol-sensing domain protein localised at the apical membrane in ectodermal epithelial cells that could act in exocytosis"

Hindelang C, Michaux G, Quintin S, Labouesse M, Gansmuller A

[European Worm Meeting, 2000]

We are interested in identifying genes implicated in the differentiation of epithelial cells in C. elegans. Epidermal cells and support cells are epithelial or epithelial-like. We have previously shown that the lin-26 gene specifies and/or maintains the fate of ectodermal epithelial cells. In particular the weak lin-26(n156) allele affects the structure of most support cells. Because che-14(e1960) has been reported to show similar defects (Perkins et al., Dev. Biol. 56: 110-156, 1986), we decided to study the che-14 gene. We isolated two new che-14 mutations and the KO consortium provided a null allele. All these mutations appear to result in a similar phenotype which is characterised by partial lethality and a dye-filling defect. The lethality affects mainly the larvae and adults: the dead worms resemble rods filled with fluid, and always have abnormalities in the excretory canal. Electron microscopy reveals that the dye-filling phenotype is due to the closure of the amphid canal and that dark vesicles accumulate in the cytoplasm of socket and sheath cells. Preliminary electron microscopy studies also indicate that the structure of the excretory canal is abnormal. We cloned the che-14 gene which encodes a multipass transmembrane protein homologue of Disp, a Drosophilaprotein dedicated to the release of cholesterol-modified Hedgehog from signaling cells. CHE-14 also displays similarities with Patched, the Hedgehog receptor, and NPC1, a protein implicated in vesicle trafficking between late endosomes and lysosomes and responsible for the Niemann-Pick disease. All of these proteins are thought to have 12 transmembrane segments and a Sterol-Sensing Domain and therefore they define a new family of transmembrane proteins. To further analyse the CHE-14 function, we decided to use a che-14::gfp construct which rescues the Che-14 phenotype. The CHE-14::GFP fusion protein is expressed in almost all ectodermal epithelial cells and is mainly localised to the apical membrane in seam cells, the vulva, the anus, and in support cells for which the apical side corresponds to the channel formed by these cells at the tip of the nose. By deletions in this construct, we found that the carboxy-terminal does not seem to have any role, and that any deletion bigger than 40 aa or deleting transmembrane domains leads to a non-functional protein that fails to localise to the apical membrane. To find the molecular basis for the similarity of lin-26(n156) and che-14(e1960) phenotypes, we looked at che-14 expression in the lin-26 null mutant. In lin-26(mc15), the che-14 expression is normal. However after lin-26 overexpression, che-14 becomes ubiquitously expressed in early embryos. We conclude that i) there is probably a redundancy between lin-26 and one or more other genes for the control of che-14 and ii) that lin-26 directly or indirectly controls che-14 expression. Based on i) the CHE-14 apical localisation, ii) the accumulation of vesicles near the apical side of the amphid support cells, and iii) the function of homologous proteins, we propose that CHE-14 may have a role in apical exocytosis in ectodermal epithelial cells.

Labouesse M et al. (2000) European Worm Meeting "LET-413, a basolateral protein with leucine-rich repeats and one PDZ domain, is required for the assembly of adherens junctions in C. elegans"

Bosher JM, Baillie DL, Legouis R, Labouesse M, Gansmuller A

[European Worm Meeting, 2000]

Epithelial cells are characterised by apical-basal polarity, a property which relies on different protein and lipid contents in the apical and basolateral compartments. Tight and adherens junctions demarcate the apical compartment and ensure epithelial integrity. Proper morphogenesis of these junctions and correct establishment of epithelial polarity are critical for normal development and histogenesis. However, the mechanisms that control these processes are poorly understood. We previously performed a deficiency screen using the mAb MH27 to identify loci required for embryonic morphogenesis (1). In sDf35 embryos adherens junctions are discontinuous or in many areas completely absent and the embryos arrest at the 1,5-fold stage,eventually rupturing ventrally. We found that embryos homozygous for any of the four mutations affecting the let-413 gene show the same phenotype as sDf35 embryos. We have cloned the let-413 gene which encodes a member of a novel class of proteins with two notable features: the presence of 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases (C. elegansSOC-2/SUR-8), and one PDZ domain similar to the PDZ domain of proteins that localise receptors to the membrane (PSD-95, chapsyn-110). The LET-413 protein localises to the basolateral membrane of epithelial cells. Immunostaining and electron microscopy analyses of let-413 mutants revealed that the integrity of adherens junctions is disrupted, leading to a loss of cell polarity (apical markers are not localised properly), severe morphogenetic defects and embryonic lethality. Moreover, the actin cytoskeleton of epithelial cells is completely disorganised in let-413 mutants. We suggest that LET-413 acts as an adapter protein involved in polarising protein trafficking in epithelial cells. (1) Labouesse, M. Dev Dyn 210, 19-32 (1997).

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.