Dell M et al. (1998) West Coast Worm Meeting "unc-34 IS enabled. I GUARANTEE IT"

Dell M, Mickey KM, Garriga G, Bloom L, Gertler F, Forrester WC, Horvitz HR

[West Coast Worm Meeting, 1998]

unc-34 is required for cell and neuronal growth cone migrations; unc-34 mutations disrupt nearly all cell and growth cone migrations that have been analyzed. In particular, unc-34 mutations affect the cell migrations of the HSN, CAN, ccM, ALM, and Q cell neuroblasts as well as the growth cone migrations of the HSN, CAN, VD and DD axons. In addition, unc-34 mutants also show a general disorganization of axon bundles of the ventral nerve cord. To address the role of unc-34 in these migrations we cloned the gene. unc-34 maps to a cosmid gap on the left arm of chromosome V, approximately one m.u. to the left of unc-60. A BLAST search using the sequence of the Drosophila gene enabled (ena) detected a C. elegans homologue from a YAC sequence that covers the cosmid gap in this region. Ena is conserved from flies to mammals and has been implicated in cytoskeletal dynamics and stability through F-actin assembly; the murine homologue, mEna localizes to the cytoskeleton and binds profilin. Mutations in Drosophila ena cause defects in the CNS and PNS, including misrouted axons and disorganized axon bundles. The role of ena in cell and neuronal growth cone migrations, and the presence of an ena homologue in the unc-34 region, suggested to us that unc-34 may encode the C. elegans homologue of enabled. Three results showed that unc-34 is indeed an ena homologue. First, RNA interference with C. elegans ena sequences produced an unc-34-like phenotype. Second, genomic Southern blots of DNA from unc-34 mutants showed that the ena homologue was deleted in unc-34(e951) mutants. Third, sequencing the 5' end of the ena gene from unc-34 mutants showed that unc-34(gm104) is an amber stop early in the gene and unc-34(gm114) is an alanine to threonine mutation. Our work also demonstrates that, in addition to the requirements for Ena in nerve bundle formation seen in Drosophila, Ena plays a significant role in cell migration. We are currently investigating the function of C. elegans abl and dab, two other genes in the Drosophila enabled pathway.

J Grabitzki et al. (2004) European Worm Meeting "THE PCOME OF CAENORHABDITIS ELEGANS IDENTIFICATION OF PHOSPHORYLCHOLINE-SUBSTITUTED PROTEINS AND COMPARISON OF DEVELOPMENTAL CHANGES IN THE EXPRESSION OF THIS EPITOPE"

R Geyer, G Lochnit, J Grabitzki

[European Worm Meeting, 2004]

Caenorhabditis elegans has been found to be good model system for parasitic nematodes, drug screening and developmental studies. Like the respective parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins, carrying, in part, phosphorylcholine (PC) substitutents, which might play important roles in nematode development, fertility and, at least in the case of parasites, the survival within the host (1). With the exception of a major secretory/ excretory product from Achanthocheilonema viteae (ES-62) (2) and the aspartyl-protease ASP-6 (3), no other proteins carrying this epitope has been identified and studied in detail yet. For C. elegans two N-linked PC-epitopes have been reported so far: (I) a pentamannosyl-core structure carrying three PC-residues (4) and (II) a trimannosyl-core species elongated by a N-acetylglucosamine substituted at C-6 with PC (5). Furthermore, in Dauer larvae of C. elegans there was evidence for the presence of glycans with the composition PC1Hex3HexNAc3 to PC2dHex2Hex4HexNAc7 (6). Here we present the 2D-electrophoretic separation of C. elegans proteins, the comparison of the PC-substitution pattern in distinct developmental stages and the mass spectrometric identification of PC-modified proteins. References: 1.Lochnit, G., Dennis, R. D., and Geyer, R. (2000) Biol Chem 381, 839-847 2.Harnett, W., Harnett, M. M., and Byron, O. (2003) Curr Protein Pept Sci 4, 59-71 3.Lochnit, G., Grabitzki, J., Henkel, B., and Geyer, R. (2003) Biochemical Journal submitted 4.Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002) J Biol Chem 277, 49143-49157 5.Haslam, S. M., Gems, D., Morris, H. R., and Dell, A. (2002) Biochem. Soc. Symp. 69, 117-134 6.Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004) Proc Natl Acad Sci U S A 101, 3404-3408

N Hawkins et al. (1999) International C. elegans Meeting "WASP and Disabled work in parallel to UNC-34, a C. elegans homolog of enabled"

G Garriga, E Kong, W Forrester, N Hawkins

[International C. elegans Meeting, 1999]

Mutations in unc-34 partially disrupt the migrations of cells and axon growth cones and the fasciculation of axons into nerve bundles. We have found that unc-34 encodes a homolog of the Drosophila Enabled (Ena) and mouse Mena proteins that have been implicated in regulating cytoskeletal dynamics though F-actin assembly (Dell et al. 1998 WCWM Abstract). In Drosophila , ena is necessary for axon outgrowth and fasciculation. Our results show that in C. elegans Ena also functions in cell migration. The weak effects of unc-34 null mutations on cell and growth cone migrations suggest that additional genes act to regulate actin assembly. In RNAi experiments, we have found that a gene similar to Drosophila disabled ( dab ) and a gene encoding a homolog of the Wiskcott-Aldridge Syndrome Protein (WASP) act in parallel to unc-34 to regulate cell motility. In Drosophila , Disabled (Dab) is a negative regulator of Ena. In C. elegans , in contrast, we have found that a dab -like gene appears to act in parallel to unc-34 . RNAi experiments with dab result in cell migration defects, and dab(RNAi) in an unc-34 null mutant results in cell migration defects that are more severe than in either an unc-34 or dab(RNAi) background. WASP has also been implicated in cytoskeletal dynamics. In addition, Ena and WASP have an EVH1 domain at their amino termini which binds vinculin and zyxin. wasp(RNAi) animals display no obvious phenotypes, but wasp(RNAi) in unc-34 null mutants results in synthetic lethality. Most animals die as embryos, and the few embryos that hatched were too disorganized to score the positions of migratory cells. To test whether wasp was involved in cell motility, we injected wasp RNA into unc-34(e315 ts ) . At the permissive temperature many of the embryos hatched and these animals were found to have severe cell migration defects. We are currently screening for mutations that are synthetically lethal with unc-34 .

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

Gallegos ME et al. (2000) West Coast Worm Meeting "sax-1 and sax-2 act in parallel with unc-34 to Maintain Neuron Polarity."

Zallen JA, Bargmann CI, Gallegos ME

[West Coast Worm Meeting, 2000]

During development each neuron in C. elegans extends a precise number of processes from its cell body. This polarity is established normally in sax-1 and sax-2 mutants, but later in development ectopic neurites often emerge from the soma of select neurons. In addition, sax-1 and sax-2 mutants have neurons with enlarged, irregularly shaped cell bodies (Zallen et al. 1999). Whereas similar phenotypes have been seen in a variety of mutants known to disrupt neuron function, neurons in sax-1 and sax-2 mutants function normally suggesting that sax-1 and sax-2 may act more directly with the actin cytoskeleton to maintain neuron polarity. To ask if the late-emerging neurites in sax-1 and sax-2 mutants result from a deregulated axon guidance pathway, I made double mutants of sax-1 and sax-2 with various axon guidance mutants including unc-34, a homolog of Enabled (M. Dell and G. Garriga, pers. comm.). unc-34 lf mutants have axon guidance and early axon termination defects suggesting that unc-34 functions to direct axon guidance and promote axon outgrowth during development. Surprisingly, unc-34;sax-1 and unc-34;sax-2 double mutants are enhanced for the ectopic neurite outgrowth defect. Furthermore, these ectopic neurites emerge after initial polarity has been established. These results suggest that sax-1 and sax-2 act in parallel with unc-34 to inhibit ectopic neurite outgrowth. Since the early axon termination defects of unc-34 suggest that it functions to promote axon outgrowth one interesting possibility is that unc-34 is bifunctional. These results also demonstrate that unc-34 plays a role during the establishment and maintenance of neuron polarity. sax-1 encodes a S/T kinase related to the Ndr protein kinase in humans (62% id.) and flies (60 % id.) (Zallen and Bargmann submitted). While the function of the Ndr kinases is unknown, other closely related kinases have been shown to play a role in cell shape and polarity in nonneuronal cells. Since double mutant analysis places sax-1 and sax-2 in the same genetic pathway, I am in the process of cloning sax-2 in order to identify other components of this kinase pathway. Progress in cloning and additional phenotypic characterization will be presented.

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.