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.

Tinley TL et al. (1998) West Coast Worm Meeting "UNC-96: FINDING A NEEDLE IN A HAYSTACK"

Tinley TL, Tang XX, Mercer KB, Santoianni B, McClure MA, Benian GM, Alberico C

[West Coast Worm Meeting, 1998]

A single mutant allele of unc-96, su151, was originally identified by Zengel and Epstein and a second allele, r291, was later isolated in Phil Anderson's lab. We have since identified additional alleles by a non-complementation screen. All alleles show normal movement but have a characteristically disorganized body wall muscle structure. By polarized light microscopy, there are highly birefringent "needles" near the ends of muscle cells, without any definite A or I bands. By electron microscopy, the most severe allele, su151, has extra collections of thin filaments or intermediate filaments, and masses of thick and thin filaments in abnormal locations. After more finely mapping unc-96, we inspected the genomic sequence for intermediate filament (IF) protein candidates using ACeDB. One candidate, homologous to Intermediate Filament Protein A, a muscle specific IF isoform purified from the nematode Ascaris, is contained in cosmids M6 and T19D7. Using either these two overlapping cosmids or a 12 kb fragment predicted to contain the entire gene, we were able to achieve transformation rescue of unc-96. Previously, Dodemont et al. (EMBO J. 13: 2625, 1994) characterized 8 genes from C. elegans that encode IF proteins. We find that unc-96 corresponds to their "c2" gene which they had shown detects four mRNAs on a northern blot. With the advantage of the genome sequence, we have confirmed, by RT-PCR, 10 more exons which add ~640 residues to the N-terminus. We are in the process of confirming additional 3' exons, as well. We are also developing antibodies that will help us distinguish the many different products of this gene. The IF cytoskeleton of mature striated vertebrate muscle is composed primarily of the muscle specific IF protein desmin. Desmin knock-out mice form muscle of normal structure, but soon after birth, the myofilament lattice begins to become disorganized and then degenerates in proportion to muscle usage. To address the possibility that the disorganization of unc-96 is due to deterioration of the lattice as a result of contraction, we made a double mutant of unc-96 with a mutation in the major myosin heavy chain that decreases myosin activity but does not appreciably affect muscle structure. unc-54(s95) is a missense mutation in the head of the major myosin heavy chain near the ATP binding site (Dibb et al., J. Mol. Biol. 183: 543, 1985). unc-54(s95); unc-96(su151) shows a remarkable increase in organization, and the needles are barely distinguishable, when present. This protection is also observed in unc-54(s95) / +; unc-96(su151).

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.

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.

DS Sieburth et al. (1999) International C. elegans Meeting "A PP2A regulatory subunit positively regulates Ras-mediated signaling during C. elegans vulval induction"

M Sundaram, DS Sieburth, M Han

[International C. elegans Meeting, 1999]

We describe evidence that a regulatory B subunit of PP2A (PP2A-B), encoded by the sur-6 gene, positively regulates a RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although there is no phenotype on an otherwise wild type background, loss of sur-6 PP2A-B function suppresses the phenotype caused by an activated Ras mutation. Furthermore a loss-of-function sur-6 PP2A-B mutation enhances vulval induction defects associated with mutations in lin-45 raf, sur-8 or mpk-1 MAP kinase. However, a sur-6 PP2A-B mutation, unlike a sur-8 mutation, fails to enhance vulval cell fate specification defects in a ksr-1 mutant background. Genetic epistasis analysis suggests that, like ksr-1, sur-6 PP2A-B acts downstream of or in parallel to ras and upstream of raf . Interfering with PP2A catalytic subunit expression has the same qualitative effect on ras signaling as sur-6 mutations, suggesting that PP2A has a positive regulatory role in ras -mediated signal transduction. Therefore, we propose that SUR-6 PP2A-B stimulates Ras pathway signaling by specifically regulating PP2A catalytic function and that PP2A acts together with KSR-1 to possibly up-regulate LIN-45 Raf activity.

Yukiko Yonezawa et al. (2007) International Worm Meeting "Role of the type-b insulin-like genes on larval diapause and adult lifespan in Caenorhabditis elegans."

Yukiko Yonezawa, Yasuo Kimura, Kenji Gouda, Tsuyoshi Kawano

[International Worm Meeting, 2007]

In Caenorhabditis elegans, the insulin/insulin-like growth factor-I signaling (IIS) pathway regulates larval diapause, adult lifespan, fat metabolism, and stress-resistance. C. elegans has 38 putative insulin-like peptides which are grouped into type-<font face=symbol>a</font> , -<font face=symbol>b</font> , and -<font face=symbol>g</font> according to their predicted disulfide bond pattern.1) The type-<font face=symbol>g</font> peptides have the same pattern as mammalian insulin family peptides. On the other hand, the type-<font face=symbol>b</font> peptides have one additional disulfide bond to the canonical three. In 2003, Murphy and co-workers reported that ins-7 categorized into the type-<font face=symbol>b</font> has relevancy on regulation of dauer larva formation and adult lifespan.2) This fact suggested that other type-<font face=symbol>b</font> genes also have physiological functions. Therefore, we attempted to investigate all of the type-<font face=symbol>b</font> genes by RNAi knockdown and/or gene-disruption. In this meeting, we will show positive/nagative regulation of larval diapause and/or adult lifespan by the type-<font face=symbol>b</font> genes. 1) Pierce, S. B., et al. (2001) Genes Dev. 15:672. 2) Murphy, C. T. et al. (2003) Nature 424:277.