Clites, Benjamin et al. (2021) International Worm Meeting "Genetic architecture of alcohol sensitivity in C. elegans"

Andersen, Erik, Pierce, Jonathan, Palacios, Bridgitte, Clites, Benjamin, Evans, Kathryn

[International Worm Meeting, 2021]

Developing medical treatments for alcohol use disorders (AUD) may benefit from identifying molecules that underlie behavioral responses to alcohol. Forward genetics and candidate gene screens using C. elegans have identified molecules required for behavioral responses to alcohol, including the BK potassium channel, which is a direct target of alcohol, as well as the CRF and SWI/SNF pathways, which modify alcohol responses. These and other molecules identified in C. elegans appear to work in conserved ways in mammalian models of AUD and humans. Despite their success, genetic screens likely miss other genes of large effect if, for example, those genes are essential for development, functionally redundant, or are rare. By leveraging natural phenotypic and genetic variation in 200 wild isolates from across the globe, rather than mutagenesis using the lab strain N2, we uncovered new conserved genes that contribute to alcohol sensitivity in C. elegans. Our genome-wide association study revealed four significant quantitative trait loci (QTLs), three of which have never been implicated in alcohol response in nematodes or mammals, as well as the BK channel gene, slo-1, which validated our approach. Candidate QTL genes included an FBOXA gene located within an expanded cluster of other FBOX genes, an essential nuclear transporter gene, and a rare isoform-specific allele of protein kinase D (dkf-2). Additionally, we found that deletion of dkf-2 in an N2 background conferred high resistance to intoxication. One extremely resistant wild isolate, JU830, which harbors all resistant genotypes at the QTLs, was six standard deviations above mean resistance. In contrast to the small indel and SNP variation associated with the four QTL in other strains, closer inspection of the BK channel slo-1 locus in JU830 identified large indels in a number of its introns as well as its 5'UTR. Our results suggest that C. elegans underwent selection for alcohol sensitivity and resistance in nature. The three novel genes identified here, especially the protein kinase D, might represent druggable targets for novel treatments for alcohol addiction and abuse.

Podbilewicz B (1994) Worm Breeder's Gazette "What Snakes and Worms Have In Common: Disintegrins"

Podbilewicz B

[Worm Breeder's Gazette, 1994]

WHAT SNAKES AND WORMS HAVE IN COMMON: DISINTEGRINS Benjamin Podbilewicz, MRC Laboratory of Molecular Biology, Cambridge, UK

Rubenzer, Kaelin et al. (2021) International Worm Meeting "Identifying genes that contribute to social defects in autism using wild-isolate C. elegans"

Clites, Benjamin, Williams, Courtney, Pierce, Jonathan, Rubenzer, Kaelin, Andersen, Erik

[International Worm Meeting, 2021]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder often characterized by significant problems with social skills. Human genetic studies have associated ASD with mutations in over 200 genes; however, it is unknown which ASD candidate genes are causal versus coincidentally associated with social deficits. Testing for causality of ASD-related phenotypes in such a large list of genes is challenging, especially when one considers that expressivity of ASD phenotypes may depend on mutations across multiple genes. We sought to leverage the convenient behavioral genetics of Caenorhabditis elegans to test for social deficits in orthologs of ASD-associated genes. C. elegans displays social behaviors through social feeding and clumping. The common wild-type lab strain N2 displays weak social behavior due to a background mutation in the neuropeptide-Y-like receptor, npr-1, but most natural wild strains are strongly social. We evaluated levels of social behaviors of 24 wild strains that harbor unique, naturally occurring variants predicted to alter function of conserved ASD candidate genes. We discovered that strains with predicted change-of-function mutations in ASD- implicated genes demonstrated significantly lower levels of social behavior compared to control social strains without these mutations. These genes include orthologs of human genes BCL11A, PDCD2, SHANK3, STX1A, NLGN1, IL-17, IL17R, CHD2, and SETD5. In addition to finding strains with dual defects in bordering and clumping, intriguingly, we identified strains that exhibited selective defects in one of the two social behaviors suggesting that they are genetically separable. We also analyzed the evolutionary conservation of ASD-implicated genes to characterize critical regions for naturally occurring variations. Identification of genes and their critical regions that may predict ASD phenotypes when mutated could help medical professionals and genetic counselors identify at-risk families, while also providing useful insight to explain causality in the biological foundations of ASD-related phenotypes.

Chimal-Monroy JDS et al. (2020) Int J Dev Biol "Cell fusion and fusogens - an interview with Benjamin Podbilewicz."

Escalante-Alcalde DM, Chimal-Monroy JDS

[Int J Dev Biol, 2020]

Cell fusion is a process in which cells unite their membranes and cytoplasm and is fundamental for sexual reproduction and embryonic development. Among the best-known cell fusion processes during animal development are fertilization, myoblast fusion, osteoclast generation, and vulva formation in Caenorhabditis elegans. Although it is involved in many other functions in unicellular and multicellular organisms, little is known about the mechanisms of cell fusion and the genes that code for the proteins participating in this process. Benjamin Podbilewicz has dedicated many years to understanding the process and mechanisms of cell fusion. In this interview, he spoke to us about how he began his studies of this process, his contributions to this exciting field, scientific ties with Ibero-America and his strategies for a well-balanced scientific/personal life.

Uccelletti D et al. (2008) Mol Biol Cell "APY-1, a novel Caenorhabditis elegans apyrase involved in unfolded protein response signalling and stress responses."

Palleschi C, Alberti A, Farina F, Mancini P, Hirschberg CB, Uccelletti D, Pascoli A

[Mol Biol Cell, 2008]

Monitoring Editor: Benjamin Glick Protein glycosylation modulates a wide variety of intracellular events and dysfunction of the glycosylation pathway has been reported in a variety of human pathologies. Endo-apyrases have been suggested to have critical roles in protein glycosylation and sugar metabolism. However, deciphering the physiological relevance of Endo-apyrases activity has actually proved difficult, owing to their complexity and the functional redundancy within the family. We report here that an UDP/GDPase, homologous to the human apyrase Scan-1, is present in the membranes of Caenorhabditis elegans, encoded by the ORF F08C6.6 and hereinafter-named APY-1. We showed that ER stress induced by tunicamycin or high temperature resulted in increased transcription of apy-1. This increase was not observed in C.elegans mutants defective in ire-1 or atf-6, demonstrating the requirement of both ER stress sensors for up-regulation of apy-1. Depletion of APY-1 resulted in constitutively activated UPR. Defects in the pharynx, and impaired organization of thin fibers in muscle cells, were observed in adult worms depleted of APY-1. Some of the apy-1(RNAi) phenotypes are suggestive of premature aging, since these animals also showed accumulation of lipofuscin and reduced lifespan that was not dependent on the functioning of DAF-2, the receptor of the insulin/IGF-1 signaling pathway.

Larsen MK et al. (2006) Mol Biol Cell "MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans."

Tuck S, Larsen MK, Faergeman NJ, Knudsen J

[Mol Biol Cell, 2006]

Monitoring Editor: Benjamin Glick The budding and fission of vesicles during membrane trafficking requires many proteins including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and what precisely their roles are in vesicle biogenesis is not yet understood. Here we present the first report on MAA-1, a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in C. elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway, and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Whereas in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.

Benjamin Schlager et al. (2006) European Worm Meeting "An Ancestral Module Patterns a Derived Nematode Vulva Equivalence Group"

Ralf Sommer., Benjamin Schlager

[European Worm Meeting, 2006]

Benjamin Schlager and Ralf Sommer. Nematode vulva formation provides a paradigm to study the evolution of pattern formation and cell fate specification. The Caenorhabditis elegans vulva is generated from three of six equipotent cells that form the so-called vulva equivalence group. During evolution, the size of the vulva equivalence group has changed: Panagrellus redivivus has eight, C. elegans six and Pristionchus pacificus only three cells that are competent to form vulval tissue. In P. pacificus, the reduction of the vulva equivalence group is achieved by programmed cell death of individual precursor cells. We have identified the genes controlling this cell death event and the molecular mechanism of the reduction of the vulva equivalence group. Mutations in Ppa-hairy, a gene which is unknown from C. elegans, result in the survival of two precursor cells, which expands the vulva equivalence group. Mutations in Ppa-groucho cause a similar phenotype. Ppa-HAIRY and Ppa-GROUCHO form a molecular module that represses the Hox gene Ppa-lin-39 and thereby, reduces the size of the vulva equivalence group. The C. elegans genome does not encode a similar hairy-like gene and no typical HAIRY/GROUCHO module exists. Instead, the size of the C. elegans vulva equivalence group is regulated by the EGF and WNT signaling pathways.. Therefore the derived vulva equivalence group of P. pacificus is patterned by an ancestral hairy/groucho module.. I will also present a highly speculative model of what kind of adaptive values might be associated with the Pristionchus specific reduction of the size of the vulva equivalence group.

Kariya K-i et al. (1997) Worm Breeder's Gazette "C. elegans homologs of ralGDS, AF-6, Cdc25 and phospholipase Cb interact with Let-60"

Barstead RJ, Watari Y, Shibatohge M, Kariya K-i, Goshima M, Kataoka T, Liao Y

[Worm Breeder's Gazette, 1997]

Mammalian Ras proteins regulate multiple effector molecules. They include Raf family (Raf-1, B-Raf, A-Raf), RalGDS family (RalGDS, RGL, RGF) and AF-6. C. elegans genetic studies contributed a lot to understanding of Ras function, especially regulation of Raf-MEK-MAPK pathway. However, studies have been limited to Raf and its downstream. Our long-term goal is to understand all pathways regulated by C. elegans Let-60. Here we report progress of our search for new Let-60 effectors and genetic study of their functions. We first screened for Let-60-binding proteins by using the yeast two-hybrid system. lACT-RB2, a random-primed mixed-stage C. elegans cDNA library was constructed in lACT that express cDNA clones fused to GAL4 activator domain. After conversion to the plasmid form (pACT-RB2), it was co-transformed into the test yeast strain CG-1945 with a plasmid pAS2-1-Let-60V12, expressing an activated Let-60 mutant as a fusion with GAL4 DNA-binding domain. After screening of approximately 106 transformants, 88 colonies were identified as both His+ and LacZ+. Plasmid clones were recovered and confirmed to confer both His+ and LacZ+ phenotypes when co-transformed again with pAS2-1-Let-60V12 but not when co-transformed with pAS2-1 vector. Inserts of confirmed clones were characterized by DNA sequencing. The sequencing data were used to identify cosmid clones containing overlapping genomic sequences by the BLASTN search (thanks to the C. elegans genome sequence project). The cDNA sequences together with the cosmid sequences were also used to identify EST clones containing overlapping sequences (thanks to Dr. Y. Kohara at NIG, Japan). When corresponding cosmid was not found, the cDNA sequences or the EST sequences were used to identify homologous proteins from the protein database by the BLASTX search. The clones comprised 6 classes. They included those encoding C. elegans Raf (Ce-Raf, 35 clones), C.elegans homologs of RalGDS (Ce-RalGDS, 29 clones), of AF-6 (Ce-AF-6, 6 clones), of Cdc25 (Ce-Cdc25, 10 clones) and of phospholipase Cb (Ce-PLCb, 2 clones), and those encoding other proteins (6 clones). Since lACT-RB2 represents 107 independent clones, this screen is not saturated yet. Predicted structures of newly found Let-60 effectors are shown in Fig. 1. We found Ce-RalGDS (F28B4.2) initially by simply performing a BLASTP search with mouse RalGDSA. Two-hybrid clones contained regions encoding the middle Cdc25 homology domain, the C-terminal Ras-interacting domain and a termination codon. pACT-RB2 plasmid library was used as a template to obtain sequence for N-terminal portion by PCR with a SL1 splice leader primer and a cDNA-specific primer. Clones for Ce-AF-6 contained the initiation codon and regions encoding the N-terminal Ras-interacting domain and the middle GLGF/DHR motif (no cosmid). Sequence for the C-terminal portion and the termination codon was found in a EST clone. Clones representing Ce-Cdc25 (T14G10.2/K04D7) encoded a Cdc25 homology domain most similar to that of Cdc25Mm/RasGRF. But regions around this domain were divergent from Cdc25Mm/RasGRF. It is possible that Ce-Cdc25 is a downstream effector of Ras, not a homolog of Cdc25Mm/RasGRF, an upstream regulator of Ras. Clones for Ce-PLCb (F31B12.1) encoded the X, Y and C2 domains found in the human counterpart. The Ras-binding domain was mapped to the C-terminal 300 amino acids by deletion analysis. F31B12.1 contains a very long N-terminal extension not found in human PLCb. But this prediction seems to be correct since PCR with a primer containing the predicted initiation codon and a downstream primer using the pACT-RB2 plasmid library gave a band with a predicted size. By computer analysis, Ponting and Benjamin recently proposed the existence of a family of Ras-associating domains (RA domain) including those of RalGDS and AF-6 [1]. Their list of proteins containing the RA consensus included Ce-RalGDS, Ce-Cdc25 and Ce-PLCb. However, Ce-AF-6 was not detected in their search, since it was not contained in any sequenced cosmids. Also, the list did not include Cdc25Mm/RasGRF, supporting the uniqueness of Ce-Cdc25. Search is in progress for mutant worms carrying Tc1-insertions within these genes. A PCR-based method was used to screen 108 plates, each started with ten MT3126 worms (thanks to the CGC). Two alleles each for Ce-RalGDS and Ce-PLCb were detected (we also found one allele for the previously reported Ach-1 gene[2]). After sib-selection, single worms were identified for all of them (thanks to Dr. Y. Andachi and the Japan C. elegans laboratory course at NIG). We are in the process of screening for worms carrying deletion of these genes by Tc1-excision.