G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Keith Nykamp et al. (2006) Development & Evolution Meeting "The Conserved La Motif Protein, LARP-1, Negatively Regulates MAPK Signalling in the Germ Line"

Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2006]

The human La protein was first described as an autoantigen present in the sera of systemic lupus erythematosus (SLE) and Sjogren's patients. Since then, a large family of La-related proteins has been described (Wolin & Cedervall, 2002). La family members are all characterized by a highly conserved La Motif (LaM), which has RNA-binding activity. Typically, LaM family members, including human La and yeast Lhp1p, harbor additional RNA Recognition Motifs (RRMs) and localize predominately to the nucleus. Both La and Lhp1p bind RNA polymerase III transcripts (i.e. tRNAs, 5S rRNAs and snRNAs) and direct their maturation. In contrast, two atypical yeast LaM proteins, Sro9p and Slf1p, do not have any other recognizable RNA-binding domains and are predominately cytoplasmic. Sro9p and Slf1p are believed to play a role in mRNA metabolism, although it is unclear what their RNA targets and exact mechanism of control might be (Sobel and Wolin, 1999). The Caenorhabditis elegans genome encodes three LaM family members: C44E4.4, T12F5.5 and R144.7/larp-1. Of the three, LARP-1 is most closely related to yeast Sro9p and Slf1p. Initial RNAi experiments indicated that LARP-1 might be important for oogenesis. To confirm the RNAi results, we isolated larp-1(q783), a deletion mutant that eliminates expression of LARP-1 protein. We find that larp-1(q783) hermaphrodites are self-fertile, but have more developing oocytes in the proximal gonad than normal. We have also found active MAP kinase to be more abundant in larp-1(q783) germ lines than in wild-type. Our working model is that LARP-1 negatively regulates MAPK activity to ensure proper oogenesis.

Fluet A et al. (1998) West Coast Worm Meeting "AGGREGATION AND TOXICITY OF HUMAN b-AMYLOID PEPTIDE VARIANTS EXPRESSED IN C. ELEGANS"

Fluet A, Johnson CJ, Fay DS, Link CD

[West Coast Worm Meeting, 1998]

We have engineered transgenic C. elegans that express the human b-amyloid peptide, which appears to play a central role in the pathology of Alzheimer's disease. As described previously, these worms, which express the peptide under the control of the unc-54 promoter, are unhealthy, have larval vacuoles, and exhibit a progressive paralysis. To better understand the molecular mechanism of b peptide toxicity, we would like to determine which, if any, of these phenotypes are specific to the expression of b peptide, and which are non-specific effects resulting from overexpression of a foreign peptide. To address this question we have sought to develop non-toxic b peptide variants, which should 'unmask' the non-specific phenotypes. We have specifically attempted to design b peptide variants that are incapable of aggregating into amyloid fibers, which have previously been reported to be directly correlated with b peptide toxicity. Single amino acid changes in the b(1-42) peptide have provided us with two interesting variants that both fail to produce amyloid aggregates: L17P and M35C. Intriguingly, multiple L17P transgenic lines all show the progressive paralysis observed in strains expressing wild-type b peptide. All M35C lines, however, are significantly healthier than the strains expressing wild-type b peptide. While the L17P variant may allow us to differentiate between b peptide's ability to form aggregates and its toxicity, the M35C variant is currently being used to differentiate between specific and non-specific toxicity resulting from b peptide expression. We have also isolated a second non-aggregating version of the b peptide, the single-chain b dimer. Like the M35C variant, this version of the b peptide does not aggregate and animals that express it are healthier than those expressing the wild-type b peptide. Quantitative immunoblot analyses on these strains demonstrate that the differences we see in aggregation of the variants are due to qualitative differences in the peptides and not quantitative differences in their levels of expression. These results also suggest that the progressive paralysis phenotype is specifically due to b peptide toxicity. We are currently using the non- or less-toxic b peptide variants in conjunction with the wild-type b peptide to look for molecular correlates of b peptide toxicity. We have preliminary results from immunoblots of Hsp16 expression that suggest that this heat shock protein is upregulated in response to b amyloid expression, and that this upregulation is significantly reduced in lines expressing the non-aggregating b dimer.

Kao G et al. (1995) International C. elegans Meeting "Molecular Genetics of the Worm Laminin beta Gene."

Wadsworth WG, Kao G

[International C. elegans Meeting, 1995]

We are interested in investigating the roles that the extracellular matrix (ECM) plays during development of C. elegans. Among other things, the ECM influences cell migrations, positioning of muscle cells, morphogenesis and the regulation of developmental signals. The need to understand more about the ECM is underscored by the fact that the cell guidance cue, UNC-6, is a laminin related molecule which may interact with ECM components. The focus of this work is the laminin beta (formerly B1) protein which, as part of the heterotrimeric glycoprotein laminin, is an important part of the ECM. The worm homologue has been cloned and sequenced. It corresponds to the homozygous viable mutation lam-1(rh219). The phenotypic analysis of this mutant, which was isolated by Ed Hedgecock, reveals defects in excretory canal outgrowth, ray morphogenesis and germline development. In addition, the gonadal sheath cells are abnormal and muscles can be mispositioned. We will be presenting work on the isolation of null mutations in laminin beta, the phenotypic characterization of rh219 and the evidence for genetic interactions of lam-1 with other matrix associated proteins.

Kao G et al. (1997) International C. elegans Meeting "THE C. ELEGANS LAMININ BETA AND GAMMA SUBUNITS"

Wadsworth WG, Huang C-c, Kao G, Karantza V

[International C. elegans Meeting, 1997]

We are interested in the roles that basement membranes have during development and the mechanisms of their assembly. In particular, we have been studying laminin beta and gamma subunits. The N-terminal domains VI and V of laminin subunits are homologous to netrin UNC-6, a cell and axon guidance cue, and these domains may have important biological activities. A b subunit mutant, lam-1 (rh219) was isolated by Ed Hedgecock. By EM we find that mutants have delaminated basement membrane and abnormal tissue development, similar to epi-1 alpha subunit mutants(K.Joh and E. Hedgecock). We have found that allele rh219 contains a leucine to phenylalanine change near the end of domain VI. This leucine is conserved between fly, mouse, and human beta subunits. We have isolated and are now analyzing a second allele, lam-1(ur53). Mutations have not yet been uncovered in the gamma subunit, which has been positioned on the X chromosome by the sequencing project. We have analyzed beta and gamma subunit cDNAs. The most highly conserved domains, VI, V, and III show 50-60% amino acid identity with their fly and mouse counterparts. A consensus site for nidogen is found in gamma domain III-4 and a Ca++ binding site is found in beta domain V-3. In vertebrates many laminin isoforms are known, however only one has so far been found in C. elegans. Currently we are studying the expression of laminin subunits using GFP fusions, in situ hybridization, and antibodies.

Xu, T. et al. (2019) International Worm Meeting "How does an animal move? An integrative model of C. elegans forward locomotion."

Kawano, T., Xu, T., Po, M., Shao, S., Wu, M., Zhen, M., Huo, J., Wen, Q., Lu, Y.

[International Worm Meeting, 2019]

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with the B-type motor neurons that execute forward locomotion. We combined genetic analysis, optogenetic manipulation, calcium imaging, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generate rhythmic activity, constituting distributed oscillators. Second, AVB premotor interneurons use their electric inputs to drive bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrain the frequency of body oscillators, forcing coherent bending wave propagation. Despite substantial anatomical differences between the motor circuits of C. elegans and higher model organisms, converging principles govern coordinated locomotion.

Melissa Harrison et al. (2001) International C. elegans Meeting "FUNCTIONAL STUDIES OF THE CLASS B SYNMUVS REQUIRED FOR THE NEGATIVE REGULATION OF VULVAL INDUCTION AND CHARACTERIZATION OF A NOVEL CLASS B SYNMUV GENE"

Melissa Harrison, Bob Horvitz

[International C. elegans Meeting, 2001]

A receptor tyrosine kinase/Ras pathway necessary for vulval induction in C. elegans has been shown to be negatively regulated by two redundant pathways, defined by the synthetic Multivulva (synMuv) class A and B genes. Mutations in members of either class alone do not result in a Multivulva phenotype. However, animals containing loss-of-function mutations in both a class A and a class B gene display a synMuv phenotype. While most of the identified class A genes encode novel proteins, many of the class B synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, and let-418 Mi-2, have homologs in other organisms known to be involved in chromatin modification complexes. Three mutations that define a new class B synMuv were identified in a recent screen for additional class B genes performed with Craig Ceol and Frank Stegmeier in our laboratory. We mapped these mutations to the far left of the X chromosome and are currently attempting to clone the gene by cosmid rescue. Several mammalian homologs of the class B synMuv genes have been shown to be components of the NuRD chromatin remodelling complex. Additionally, some of the proteins in the synMuv B pathway have been shown to physically interact either in vitro (1,2) or in yeast two-hybrid assays (3). We are attempting to use co-immunoprecipitation experiments to expand the current knowledge of the presumptive class B protein complex in C. elegans . Analysis of the effects of various mutations on co-immunoprecipitation may allow us to assign functionality to some novel class B genes. Currently, there is little understanding of how the class A and B synMuvs redundantly regulate vulval induction. Class A and B synMuvs might play redundant roles in the formation of a chromatin modifying complex, and such physical interactions could also be demonstrated through co-immunoprecipitaion experiments. (1) Ceol, C. and H.R. Horvitz (in press). (2) Lu and Horvitz. (1998) Cell 95 : 981. (3) Walhout et al. (2000) Science 287 :116.

Sun, Simo et al. (2017) International Worm Meeting "Neural and molecular basis involved in low preference to Bifidobacterium infantis in Caenorhabditis elegans."

Nishikawa, Yoshikazu, Kage-Nakadai, Eriko, Sun, Simo

[International Worm Meeting, 2017]

Neural and molecular mechanisms underlying food preference have been poorly understood. We previously showed that Bifidobacterium infantis, one of the well-known probiotic bacteria, extend the lifespan of Caenorhabditis elegans (Ikeda et al., Appl. Environ. Microbiol., 2007). In this study, we found that C. elegans exhibited low preference to B. infantis when compared with a standard food E. coli. Genetic screens identified tol-1 (a sole homolog of mammalian Toll-like receptors) and daf-16/FoxO as positive regulators of low preference to B. infantis. Worms with mutations in canonical TLR signaling molecules (pmk-3, mom-4 and ikb-1) that are employed in avoidance behavior to a pathogenic bacterium Serratia marcescens (Brand et al.,Curr. Biol., 2015) showed a normal behavior to B. infantis, suggesting alternative mechanisms. Genetic analyses revealed that the function of daf-16 isoform b in AIY neurons is responsible for the behavior to B. infantis. Because daf-16b regulates the AIY neurons morphology during development (Christensen et al., Development, 2011), daf-16b may play roles in AIY development to organize low preference to B. infantis.

AI Cid et al. (1999) International C. elegans Meeting "Investigating the function of the SMA-1 SH3 domain in C. elegans morphogenesis"

AI Cid, J Austin

[International C. elegans Meeting, 1999]

Spectrin is a cytoskeletal actin binding protein made up of two subunits each of the proteins a -spectrin and b -spectrin. We have identified an unusual member of the b -spectrin family, SMA-1, that is required for proper morphogenesis of the C. elegans embryo. SMA-1 is a homolog of Drosophila b H -spectrin. Like other b -spectrins, SMA-1 and b H -spectrin contain an N-terminal actin binding domain, a series of spectrin repeats and a C-terminal pleckstrin homology domain. However, SMA-1 and b H -spectrin contain more spectrin repeats than other b -spectrins, and have an SH3 domain not found in other b -spectrins. SMA-1 and b H -spectrin also differ from other b -spectrins in their localization within the cell: b -spectrin is found laterally, while SMA-1 and b H -spectrin are found apically. SH3 domains are protein-protein interaction motifs which bind ligands containing proline-rich regions. We would like to know the function of this domain within SMA-1. The SMA-1 SH3 domain may play a number of roles in SMA-1 function: it could be responsible for SMA-1 localization within the cell, or it could interact with proteins that regulate SMA-1 function. We would like to determine if the SMA-1 SH3 domain plays a role in SMA-1's intracellular localization or more directly in its function in morphogenesis. We are using a yeast two-hybrid screen to identify SMA-1 SH3 domain ligands. We have identified several positives from our screen which we are in the process of sequencing. We also plan to do more in depth analysis of the SMA-1 SH3 domain, making specific mutations in the SMA-1 SH3 and examining loss-of-function phenotypes of potential SMA-1 SH3 ligands by RNA interference. To address whether the SH3 domain is responsible for SMA-1 protein localization we have begun to make GFP protein fusions containing the SMA-1 SH3 domain. By transforming worms with these SH3-GFP fusion constructs we will be able to determine where the SMA-1 SH3 domain localizes within the cell. With these methods we can attempt to understand what role the SMA-1 SH3 plays in C. elegans morphogenesis.

David Karow et al. (2001) International C. elegans Meeting "Guanylate Cyclase Beta 2 Homologues in C. elegans"

David Morton, Ronald E Ellis, Jennifer Chang, Scott Nicholls, Michael Marletta, Martin Hudson, David Karow

[International C. elegans Meeting, 2001]

Soluble guanylate cyclases (sGCs) catalyze the conversion of GTP to cGMP. In mammals, the most well-studied sGC is a heme-containing, heterodimer, composed of a 1 and b 1 subunits. Nitric oxide (NO) binds to the heme, which is ligated to the b 1 subunit, and stimulates the activity of the enzyme 400-fold. A b 1 homologue, b 2, has been cloned from rat and human kidney cDNA libraries. This subunit appears to be expressed in the kidney collecting duct cells, but its heme binding status, sensitivity to NO, and intracellular localization are unknown. We want to elucidate the structure and function of this protein. Analysis of the C. elegans genome predicted seven putative sGC b subunits, including five b 2 homologues, ( gcy-32 , gcy-34 , gcy-35 , gcy-36 , and gcy-37 ). We cloned these five homologues, by RT-PCR and RACE, and found that each of them contains the residues necessary for GTP binding, catalysis and heme binding. These findings suggest that the b 2 homologues in C. elegans are receptors in a cGMP-dependent signaling system. Our data are consistent with this hypothesis. Preliminary characterization of the bacterially-expressed N-terminus from gcy-35 suggests that it can bind heme. Promoter::green fluorescent protein (GFP) fusion studies localize all of the b 2 homologues to four candidate sensory neurons (URXL, URXR, AQR and PQR). These neurons are all connected to the pseudocoelom, and might play a role in fluid homeostasis. This possibility is consistent with data demonstrating that their mammalian homologue, b 2, is expressed in the kidney. We are now using transgenes to express Egl-1 in these four candidate neurons, so that we can eliminate them and elucidate their functions. Does NO activate b 2 and its homologues? Analysis of the C. elegans genome did not reveal an open reading frame for nitric oxide synthase (NOS). Furthermore, we have identified GC activity in C. elegans lysates and shown that this activity is NO-insensitive. These results suggest that b 2 and its homologues define a novel class of NO-insensitive cyclases. We are using biochemical techniques to examine heme-binding and ligand specificity in the b 2 homologues.