Joseph, B. et al. (2017) International Worm Meeting "Sibling Subtraction Method: A novel approach to identify causal mutations by WGS."

Fay, D., Joseph, B., Blouin, N.

[International Worm Meeting, 2017]

Sequencing technologies have made it possible to carry out whole genome sequencing (WGS) to identify the genetic variants present in mutant organisms. In addition, methods have been developed that simultaneously facilitate mapping of the causal mutation. Currently, two principal strategies for "mapping by sequencing" are used by C elegans researchers. In one approach, mutants are crossed to the polymorphic HA strain, which provides extensive SNP mapping information and localization of the causal mutation. This approach, however, is not highly practical for complex genetic backgrounds, such as the case with genetic suppressors, and the numerous polymorphisms present in the HA strain can alter phenotypes in ways that are unpredictable. A second method, EMS density mapping, exploits of the higher concentration of signature-EMS alterations in regions surrounding causal mutations following multiple backcrosses. The resolution of EMS density mapping, however, can be quite low and this method is not applicable for alleles that arise spontaneously or through other methods. We recently obtained ~30 strong suppressors nekl-2; nekl-3 double-mutant larval lethality. Our screens included a novel strategy using counter-selection that was enabled by the peel-1 toxin. Notably, neither the nekl-2 or nekl-3 single mutants used for this screen display phenotypes on their own, nor did the majority of obtained suppressors. In order to identify the causal mutations that suppress nekl-2; nekl-3 lethality, we designed and implemented a novel refinement strategy we call the Sibling Subtraction Method (SSM). For our approach, suppressed strains were backcrossed several times to ensure Mendelian inheritance and to partially remove background mutations. While performing the final backcross, we isolated strains that were homozygous for the suppressor mutation as well as strains that were non-suppressed (i.e, homozygous wild type at the suppressor loci). These isolates were combined to create a Suppressed DNA pool and a Non-suppressed Comparitor DNA pool, respectively. Following WGS, alignment, and filtering steps, common variants present in both the Suppressed and Comparitor pools were subtracted. This resulted in a very small number of candidate variants (~5) that caused non-synonymous coding changes in each of the suppressor strains. Notably, we were able to subtract more than 95% of background variants using this method. The bioinformatics analysis was performed using Cloudmap workflows in the Galaxy web platform, which are freely available and widely used by the C elegans field. Our strategy was validated on five suppressor strains and, most importantly, is generally applicable for all varieties of mutations and genetic backgrounds. As such, SSM largely obviates the need to use the HA strain and provides better candidate refinement than EMS density mapping methods.

Lazetic, V. et al. (2017) International Worm Meeting "A NimA-kinase pathway controls CDC-42 activity and actin organization in the epidermis."

Joseph, B., Fay, D., Lazetic, V.

[International Worm Meeting, 2017]

During molting the epidermal apical extracellular matrix (cuticle) is extensively remodeled. We have shown that the NimA-related kinases, NEKL-2/NEK8 and NEKL-3/NEK6/NEK7, as well as their conserved ankyrin-repeat partners, MLT-2/ANKS6, MLT-3/ANKS3 and MLT-4/INVS, are essential for molting. Both NEKLs and MLTs are expressed in the epidermal syncytia and are specifically required in hyp7 for normal molting. Our studies indicate that NEKLs and MLTs act primarily within two complexes composed of NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3, and that the MLTs function as scaffolds to ensure proper subcellular localization of the NEKLs. To understand how the NEKL-MLT network controls molting, we identified suppressors of nekl mutant molting defects. Our screens identified CDC-42, a highly conserved Rho-family GTPase, and its upstream regulator, KIN-25/SID-3. CDC-42 is important for the establishment of cell polarity and for organization of the actin cytoskeleton. KIN-25 is the ortholog of Activated CDC42 Kinase 1, which binds to CDC42 and inhibits its GTPase activity, thereby maintaining CDC42 in an active state. Notably, molting has been proposed to require extensive reorganization of actin within the epidermis. Specifically, actin is reorganized at each molt to form a series of circumferential bundles along the apical surface of hyp7, however, the mechanisms underlying this process are unknown. We found that inhibition of NEKL-MLT activities leads to defects in the pattern of apical actin and failure to form molting-specific actin bundles. Furthermore, expression studies indicate that components of the NEKL-MLT network colocalize with actin in specific regions of the epidermis. In addition, normal localization of CDC-42 in the epidermis depends on presence of NEKL-MLT proteins, and CDC-42 partially colocalizes with MLT-2. Our findings suggest that the NEKL-MLT network may negatively regulate CDC-42 activity and that partial inhibition of CDC-42 may thus reduce defects in nekl-mlt mutants. Interestingly, we also observed that downregulation of cdc-42 on its own can lead to molting defects, suggesting that there is a tight balance between NEKL-MLT and CDC-42 activities in the epidermis. Notably, studies in mammalian cells have implicated the MLT-4 ortholog, Inversin, in the regulation of CDC42 activity and apical actin organization. In addition, it was independently shown that the mammalian orthologs of NEKL-3, NEK6 and NEK7, physically associate with CDC42, however no functional connection between these proteins has been described. For the first time, our data provide in vivo evidence for a functional link between the NEKL-MLT network and regulation of the actin cytoskeleton through the CDC42 pathway.

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.

Bernazzani, S. M. et al. (2019) International Worm Meeting "A Disintegrin-containing Metalloprotease, ADM-2, is a suppressor of C. elegans NEK kinases."

Joseph, B. B., Bernazzani, S. M., Fay, D. S.

[International Worm Meeting, 2019]

The highly conserved NIMA-related kinases (NEKs) are thought to be involved in processes including DNA repair, ciliogenesis, and cell cycle regulation. Little is known, however, about NEK pathway components or targets. Our lab has previously shown that C. elegans NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 and their ankyrin-repeat partners MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS are required for molting and affect epidermal intracellular trafficking. We carried out a genetic screen to identify suppressors of the lethal molting defect exhibited by nekl-2; nekl-3 double mutants. Identified in this screen were two loss-of-function alleles of adm-2. ADM-2 contains a conserved disintegrin domain, zinc-binding motif, and a putative transmembrane domain, suggesting that ADM-2 is a functional metalloprotease. SH3 binding domains, which are found in some proteins involved in signaling pathways, are also present. ADM-2 most closely resembles the human meltrins (ADAMs-9, 12, and 19), which have been reported to be involved in cell adhesion, cell signaling, and are overexpressed in certain cancers. CRISPR deletion of either the ectodomain or the cytodomain resulted in significant suppression of molting defects in nekl-2; nekl-3 double mutants, suggesting both extracellular and intracellular functions of ADM-2 are relevant to its functions in molting. Targeted disruption of specific domains in the ADM-2 protein with CRISPR revealed significant suppression in mutants lacking a functional zinc-binding motif, intracellular furin cleavage site, or one of the three putative SH3 binding domains. A known binding partner of this domain in ADM-2/ADAM9 is sorting nexin 9 (SNX9), which is required for clathrin-mediated endocytosis. Notably, null lst-4 mutants are strongly hypersensitive to partial knockdown of nekl-2, mlt-2, or mlt-3 by RNAi. A C-terminal GFP fusion of ADM-2 localizes to cell membranes throughout the C. elegans nervous system. ADM-2 is also present throughout the cytoplasm of the hyp7 syncitium, the tissue in which NEKL-2 and NEKL-3 function. Furthermore, ADM-2 is observed in hyp7 nuclei and sequence analysis revealed an apparent nuclear localization signal directly adjacent to the cytoplasmic furin cleavage site, suggesting a possible role in signal transduction.

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.