Zhou, Junxiang et al. (2021) International Worm Meeting "beta-tubulin BEN-1 has a key role in regulating DLK-1 signal transduction"

Jin, Yishi, Sun, Yue, Zhou, Junxiang

[International Worm Meeting, 2021]

The conserved mitogen activated kinase kinase kinase (MAPKKK) DLKs play critical roles in neuronal process growth, synapse formation and axon regeneration. DLKs also act as sensors to mediate neuronal responses to different stresses, including microtubule (MT) disruption. However, it is not clear in which contexts DLKs sense MT stress and which signaling transduction mechanisms control the activity of DLKs to mediate specific functional outcomes. In C. elegans the DLK ortholog DLK-1 is normally expressed in most neurons at low levels. From a forward genetic screen for mutants displaying aberrant DLK-1 levels or localization, we identified a mutant that exhibited aggregated DLK-1 in neuronal soma. Using whole genome sequencing and genetic mapping, we determined that this mutation affected ben-1, one of six beta-tubulin isotypes in C. elegans. Levels of the transcription factor CEBP-1, a downstream target induced by DLK-1, were elevated in neurons of ben-1 mutants. These findings suggest that altering ben-1 function activates dlk-1, resulting in cebp-1 dependent transcription. A ben-1 transcriptional reporter is exclusively expressed in neurons. BEN-1 is known to convey the sensitivity to benomyl1, a microtubule-targeting compound used for killing parasitic nematodes and treating cancers. We found that benomyl treatment also caused neural DLK-1 aggregation, CEBP-1 elevation, as well as neuronal defects such as impaired synaptogenesis. Altogether, our findings suggest that changes in neuronal beta-tubulin BEN-1 containing MT cytoskeleton elicit specific stress responses to activate dlk-1/cebp-1 signaling. 1. Driscoll, M., Dean, E., Reilly, E., Bergholz, E., and Chalfie, M. (1989). Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity. J. Cell Biol. 109, 2993-3003.

Mello, Cecilia C. et al. (2011) International Worm Meeting "Spontaneous and UV-induced mutations in a small region of the C. elegans genome, the ben-1 locus."

Mello, Cecilia C., Fire, Andrew

[International Worm Meeting, 2011]

What is the spectrum of spontaneous mutations? How are mutation spectra altered in the presence of strong mutagens? To answer these questions, I used a powerful screen for isolating both spontaneous and induced mutations using the ben-1 gene, a non-essential beta-tubulin. Wild-type animals are sensitive to paralysis upon benomyl treatment, ben-1 mutants are resistant. This work of spontaneous and UV-induced mutations gives us a baseline for further studies. The strength of this approach is that it focuses mutations in a small region of the genome, allowing for a detailed analysis. This one locus does not offer a complete view of the genome, and I intend to expand this analysis into larger regions of the genome.

Chen, Di et al. (2011) International Worm Meeting "Deletion of the ribosomal S6 kinase in daf-2 mutants synergistically extends C. elegans lifespan."

Kapahi, Pankaj, Hubbard, Alan, Chen, Di, Melov, Simon, Goldstein, Ben

[International Worm Meeting, 2011]

The highly conserved insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways play critical roles in aging in multiple species. Mutations in DAF-2, the C. elegans insulin/IGF-1 receptor ortholog, double adult lifespan by activating the DAF-16/FOXO transcription factor. A deletion in the TOR target ribosomal S6 kinase (S6K) encoded by rsks-1 lead to a moderate lifespan extension in C. elegans. IIS and TOR act in a parallel but interactive manner to modulate growth, but whether and how they interact to determine lifespan is not known. Here we show that a daf-2 rsks-1 double mutant extends lifespan by nearly 5-fold compared to wild-type animals, and this synergistic extension of lifespan depends on DAF-16 activities. Gene expression profile studies indicate that there are large amount of genes differentially expressed in the very long-lived daf-2 rsks-1 animals. Through RNAi screens, we have identified suppressor genes that mediate the synergistic longevity phenotype of the daf-2 rsks-1 double mutant. Among these is a g regulatory subunit of the 5'-AMP-activated protein kinase (AMPK) complex. AMPK activities are elevated in the daf-2 rsks-1 mutant as indicated by increased transcription of AMPK g and phosphorylation of AMPK a. Furthermore, a deletion in the AMPK a catalytic subunit gene aak-2 also abolishes the synergistic longevity phenotype. Since AMPK directly activates DAF-16, our results indicate a positive feedback regulation of DAF-16 by DAF-2, S6K and AMPK. Therefore, our findings identify a critical role for AMPK-mediated interaction between daf-2 and rsks-1 that result in synergistic lifespan extension.

Zdraljevic, S. et al. (2019) International Worm Meeting "Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles."

Zhao, Y., McGrath, P.T., Rodriguez, B.C., Andersen, E.C., Hanel, S.R., Zdraljevic, S.

[International Worm Meeting, 2019]

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms that underlie resistance. Efforts to detect helminth BZ resistance in parasitic nematodes is focused on three variant sites in the orthologs of the ?-tubulin gene, ben-1, found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). In agreement with known mechanisms of BZ resistance, we found that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in ben-1. We identified 25 distinct, low-frequency ben-1 alleles within the C. elegans population, including many novel variants. Population genetic analyses indicate that these variants arose recently because of local selective pressures. Furthermore, we show that the common parasitic nematode ?-tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel ?-tubulin-independent genomic region that is correlated with ABZ resistance in the C. elegans population, suggesting that multiple mechanisms underlie BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for studying BZ resistance mechanisms.

Siddiqui SS et al. (1997) International C. elegans Meeting "ARMADILLO BY A DOZEN: 12 ARMADILLO REPEATS IN CeKAP-1, A KINESIN ASSOCIATED PROTEIN"

Ali Khan L, Siddiqui ZK, Siddiqui SS

[International C. elegans Meeting, 1997]

Previously we have reported cloning of the osm-3 gene, encoding a kinesin like protein (Shakir et al., 1993). OSM-3 shows high homology to the sea urchin spKRP85 and spKRP95 kinesin like proteins (Kinesin II). Wademan et al. (1996) have recently reported a non-motor kinesin associated protein KAP115 from sea urchin which co-purifies with kinesinII holoenzyme. We have isolated a cDNA clone (called CeKAP-1) from a nematode cDNA library, corresponding to the nematode homologue of the spKAP115 protein, that maps on the cosmid F08F8.3 (Wademan et al., 1996). Similar multi-meric complexes have been reported in mouse KIF3A-KIF3B-KAP3 (Hirokawa, 1996), and in Drosophila (KLP64D and KLP68D) (Goldstein, 1993). Gindhart and Goldstein (1997) noted that these KAP proteins harbor several repeats of the Drosophila armadillo motif. Indeed, the nematode CeKAP-1 when analyzed shows twelve contiguous armadillo repeats in the KAP-1 primary sequence, unlike spKAP115 protein that contains a total of 10 armadillo repeats, one repeat is separtaed from the other nine, which are tandem repeats. Since armadillo like repeats have been found in a variety of proteins which mediate intracellular signalling and protein interactions, a role for CeKAP-1 protein in regulating the cargo binding of the OSM-3/KLP-11( Kinesin II ) motor proteins, is worth examining. We are looking for mutants affected in the CeKAP-1 and KLP-11 genes which may show interactions with the osm-3 mutants.

Latheef, Sharmilah L.J. et al. (2013) International Worm Meeting "The use of C.elegans to identify novel mutations that confer benzimidazole resistance."

Latheef, Sharmilah L.J., Gilleard, John S., Stasiuk, Susan S.

[International Worm Meeting, 2013]

Parasitic nematodes are a huge threat to the livestock industry as well as human health. Livestock infections cause billions of dollars in production loss per annum whilst infections in humans are a leading cause of disability and poverty especially in the developing world. Intensive use of the limited number of anthelmintics available for the treatment of parasitic nematode infections in livestock has resulted in the widespread development of resistance in many regions of the world. The recent increase in community-wide anthelmintic treatment programs in humans in the developing world-using the same drug classes used in livestock- is raising concerns of similar emergence of resistance in human parasites. Hence, there is an urgent need to understand the causal mechanisms of resistance and develop new diagnostic tests and control measures. Benzimidazoles (BZ) are an important drug class for both human and animal parasite control. It is known that amino acid substitutions in the isotype-1 b-tubulin drug target at positions 167, 198 and 200 can prevent drug binding and lead to drug resistance in several parasitic livestock species. The major objective of this project is to identify additional novel causal mutations of BZ resistance using the natural genetic variation found in wild C.elegans populations. The effect of albendazole on the motility of 16 natural C.elegans isolates was tested and two strains were identified that had a high level of resistance compared to the N2 strain. The ben-1 locus was sequenced in these two strains and a novel mutation resulting in an Alanine to Proline substitution at amino acid position 185 was found in one strain and a frame-shift in the other. The functional significance of the identified non-synonymous polymorphisms was confirmed by genetic complementation studies with the ben-1(e1880) reference strain. Hence, we have identified naturally occurring strains with a high level of resistance to BZ drugs which leads to questions regarding the role of environmental pollution as a selective force. We've also identified a previously unreported amino acid substitution in the ben-1 locus that is capable of conferring resistance which has yet to be reported in parasitic nematodes.

Kage-Nakadai, Eriko et al. (2011) International Worm Meeting "Low-copy integration of transgenes by TMP/UV methods."

Yoshina, Sawako, Gengyo-Ando, Keiko, Kage-Nakadai, Eriko, Kobuna, Hiroyuki, Funatsu, Osamu, Mitani, Shohei, Ootori, Muneyoshi, Hori, Sayaka

[International Worm Meeting, 2011]

In Caenorhabditis elegans, transgenic strains are typically generated by injecting DNA into the germline to form high-copy extrachromosomal arrays. These transgenes are semi-stable and their expression are silenced in germline. In order to create single- or low-copy chromosomal integrated lines, methods using Mos1 transposon or microparticle bombardment have been developed. We have developed an alternative method by using TMP/UV, that produces low-copy integrations. We have successfully integrated low-copy of transgenes using positive selection with vps-45 rescue fragment / temperature sensitivity and negative selection with ben-1 rescue fragment / benomyl sensitivity. We confirmed that low-copy integrants express transgenes in germline. Currently, using this method, we are constructing a PhiC31-attBP / Cre-LoxP system.

Jason Morton et al. (2005) International Worm Meeting "Gene targeting in C. elegans using chimeric zinc finger nucleases"

Dana Carroll, Wayne Davis, Jason Morton, Erik Jorgensen

[International Worm Meeting, 2005]

We are seeking an efficient and reliable technique to create permanent targeted mutations in the nematode genome. Double-strand breaks (DSBs) in chromosomal DNA stimulate both homologous recombination and nonhomologous end joining (NHEJ) at the break site as part of the DNA repair process. Previous work has shown that proteins composed of a nonspecific endonuclease domain fused to an engineered zinc-finger DNA-binding domain (zinc finger nucleases, ZFNs) can produce targeted DSBs in Drosophila melanogaster. These DSBs produce mutations at defined locations in arbitrarily selected genes via NHEJ. A modified ectopic DNA molecule, homologous to the targeted sequence, can act as a template for directed DSB repair i.e., targeted gene replacement. Since many of the genes involved in DNA repair are conserved between D. melanogaster and C. elegans, ZFNs can also potentially produce defined genetic mutations in nematodes.As an initial step, we have used previously characterized ZFNs to target a defined extrachromosomal sequence and have been able to characterize the somatic mutations produced by successful cleavage and subsequent inaccurate DNA repair. Initial evidence indicates that the target DNA is cleaved at a high efficiency by the ZFNs, and several mutations arising from NHEJ have been identified. We have also produced ZFNs for a target in the ben-1 gene, and an assay to characterize somatic mutations at this locus is being developed. Additional studies designed to examine these repair events in lig-4 mutants, which lack a ligase thought to be central in the NHEJ process, are also underway.To circumvent germline silencing of transgenic DNA, we have generated low-copy integrants of ZFN expression constructs by biolistic transformation, and screens for germline ben-1 mutants are ongoing. In addition, we are using the FRT/Flp recombinase system with an RFP/GFP reporter to guide our efforts in stimulating germline expression of the ZFNs to achieve genuine gene targeting.

Keiko Gengyo-Ando et al. (1999) International C. elegans Meeting "An enhanced gene disruption method is suitable for reverse genetics of Caenorhabditis elegans"

Keiko Gengyo-Ando, Shohei MItani

[International C. elegans Meeting, 1999]

The genome project of the nematode Caenorhabditis elegans is completed. It is advantageous and informative to disrupt nematode genes to study their function. Recently, the deletion mutagenesis for reverse genetics in C. elegans has been often done with chemical mutagens instead of "transposon insertion and excision". However, efficiency of gene disruption method reported previously was still too low for large-scale isolation of deletion mutants (at most about one mutant in 400,000 genomes). To develop an efficient mutagenesis procedure, we first isolated mutant alleles of a single locus ben-1, a b-tubulin gene, from mutagenized worms with EMS, UV of short wavelength or various conditions of TMP plus UV of long wavelength. We isolated a total of 170 independent alleles from the ben-1 locus (77 by EMS, 2 by short-wavelength UV and 91 by TMP/UV) by phenotypic screenings as previously, and then analyzed DNA lesions of these alleles by PCR and sequencing. Although the probabilities for mutant isolation by EMS were higher than those by short-wavelength UV and TMP/UV, in contrast to previous assumption, the probabilities for detectable deletion mutations were very low among EMS-treated mutants (none out of 77 alleles). We also failed to find deletion mutants derived from short-wavelength UV-treatments. However, the probabilities for detectable deletion mutations were high among TMP/UV treated mutants (a total of 30 out of 91 alleles). Furthermore, we found that the deletion sizes depend on the concentration of TMP. Thus, hereafter we focused our efforts to TMP/UV mutagenesis. To improve the sensitivity and specificity of mutant detection we have optimized PCR protocol by using these deletion mutants. Finally, we could isolate deletion mutants about 50 to 100-folds as efficiently as before by an appropriate condition of TMP/UV mutagenesis and improved PCR protocol. This highly efficient procedure enabled us to do sib-selection of genomic DNAs derived from single-cultured mutant library. The method, optimized this way, is so efficient that isolating deletion mutants on the whole-genome scale is now realistic.

Thorpe CJ et al. (1997) International C. elegans Meeting "mom-2, a maternal gene required for specification of endoderm in the early C. elegans embryo, is a Wnt family member."

Thorpe CJ, Carter JC, Bowerman BA

[International C. elegans Meeting, 1997]

At the 4-cell stage of C. elegans embryogenesis, the P2 blastomere sends an inductive signal to its sister, EMS. There are two consequences to this interaction: 1) The mitotic spindle of EMS undergoes rotation, and 2) One daughter of EMS, called E, is programmed to produce only intestinal cells (Goldstein, 1992, 1993, 1995). In the absence of the induction, E develops like its sister, MS, producing pharyngeal tissue and body wall muscle. We have identified 16 mutations representing 5 genes,mom-1-mom-5 (mom=more mesoderm) that are required for induction of intestinal cells. For strong alleles of mom-2, 75-85% of embryos from mutant mothers fail to make gut and produce excess pharyngeal and body wall muscle cells. Lineage analysis and laser ablation experiments indicate that E is adopting MS-like identity in most mom-2 mutant embryos. Another gene that is required to distinguish the fates of E and MS is the maternal gene pop-1 (Lin et al, 1995). Mutations in pop-1 result in MS developing like E,suggesting that pop-1 is required to specify MS identity. pop-1 encodes a putative HMG box transcription factor that, in wild type embryos, accumulates to higher levels in MS than in E. In mom-2 mutant embryos, POP-1 accumulates to equal levels in the nuclei of MS and E, suggesting that one function of mom-2 is to downregulate POP-1 function in E. Mosaic analysis using isolated blastomeres (Goldstein, 1995; Shelton and Bowerman, 1996) indicates that mom-2 is required in P2 for sending the gut induction signal. Mutations in mom-2 alone do not appear to disrupt EMS spindle orientation. However, in embryos mutant for both mom-2 and mom-1, we observe defects in EMS spindle orientation, as well as more global effects on the orientation of cleavage axes and positioning of later blastomeres, suggesting that this pathway may function to regulate polarity throughout the early embryo. We have cloned mom-2, and shown that it encodes a member of the Wnt family of secreted signaling proteins. We are currently raising antibodies against MOM-2 protein to determine its localization.