Breton, Elizabeth et al. (2019) International Worm Meeting "The multiple roles of the CCT chaperonin in regulating germ line RNP granules."

Breton, Elizabeth, Sharp, Katherine, Montalbano, Andrea, Schisa, Jennifer, Yura, Tom

[International Worm Meeting, 2019]

In many animal species, oocytes arrest in meiosis until they are fertilized. Over time, the quantity and quality of oocytes decreases, resulting in reduced fertility. Our goal is to better understand the regulation and function of ribonucleoprotein (RNP) granules that assemble in meiotically-arrested or stressed oocytes. The C. elegans adult germ line is an excellent model to study RNP granule dynamics due to the dramatic cellular changes that occur in response to meiotic arrest. To identify regulators of RNP granule assembly, we performed an RNAi screen that identified the Chaperone Containing Tcp-1 (CCT) family. We and others initially identified subsets of the eight cct genes as positive regulators of assembly of oocyte RNP granules and embryonic P granules (Updike and Strome, 2009; Hubstenberger et al., 2015; Wood et al., 2016). Our recent results, however, strongly suggest that all seven cct genes tested normally inhibit the accumulation of the MEX-3 RNA-binding protein into large aggregates in arrested oocytes. Thus, we have uncovered a novel role for CCT chaperone subunits in modulating RNP granules in the C. elegans germ line. In yeast and mammalian cells, select CCT subunits have also been identified as negative regulators of stress granules and of polyglutamine aggregation. To determine if CCT acts directly on MEX-3, or indirectly via effects on folding of microtubule subunits, we examined beta-tubulin. We find beta-tubulin levels are significantly reduced by knockdown of cct-1 or cct-2. Since prefoldin delivers microtubule and actin substrates to CCT, we also investigated the effects of knocking down prefoldin subunits. We find that while beta-tubulin levels are significantly reduced, MEX-3 granules are not affected, suggesting that the effects of CCT on RNP granules in arrested oocytes do not occur via the microtubule or actin cytoskeleton and may be direct. Our preliminary data suggest CCT also inhibits heat stress-induced MEX-3 granules. We are currently exploring the effect of CCTs on other RNP granule components, and ultimately hope that gaining a better understanding of the regulation of RNP granules will lead to insights into their function in the germ line.

De Stasio E et al. (1994) Worm Breeder's Gazette "Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea."

De Stasio E, Stanislaus D, Lephoto C

[Worm Breeder's Gazette, 1994]

Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911

Goodwin EB et al. (1994) Worm Breeder's Gazette "The laf-1 Gene Functions in the 3'UTR-Mediated Translational Repression of the Sex Determination Gene, tra-2"

Kimble JE, Goodwin EB

[Worm Breeder's Gazette, 1994]

The laf-l Gene Functions in the 3'UTR-mediated Translational Repression of the Sex Determination Gene, tra-2. Elizabeth Goodwin# and Judith Kimble University of Wisconsin, Madison, WI. # Dept. of CMS Biology, Northwestern Univ. Medical School, Chicago Ill.

Pestrue, Chloe et al. (2021) International Worm Meeting "The CCT chaperonin selectively regulates phase transitions in the C. elegans germline"

Watkins, Brooklynne, Schisa, Jennifer, Elaswad, Mohamed, Pestrue, Chloe, Sharp, Katherine, Breton, Elizabeth

[International Worm Meeting, 2021]

When phase transitions of proteins are not properly regulated, proteins sometimes condense into aggregates that disrupt normal physiological function and lead to disease. Other times, condensation of RNA-binding proteins into ribonucleoprotein (RNP) granules is associated with homeostasis. The regulation of such phase transitions has been a topic of much inquiry but is still not completely understood. We are currently exploring the role of the Chaperone-Containing TCP1 (CCT) chaperonin in regulating RNP granule assembly in the C. elegans germline. Genetic screens performed by our lab and others initially identified several CCT subunits as promoters of PGL-1 granule assembly in embryos, and of MEX-3 granule assembly during extended meiotic arrest. In contrast, CCT inhibits stress granule and P-body assembly. Therefore, our goal was to carefully examine the role of CCT in regulating phase transitions of RNA-binding proteins in different developmental contexts. We used RNA interference (RNAi) to knockdown the expression of cct subunits in adult worms and imaged several GFP-tagged RNA-binding proteins using confocal microscopy. We find that in arrested oocytes in the fog-2 background, CCT inhibits the condensation of the P-body protein CGH-1. However, CCT does not appear to strongly regulate the condensation of the P-granule protein MEG-3. We are in the process of analyzing effects on additional RNA-binding proteins. We also find that in young, wild-type hermaphrodites CCT inhibits the condensation of the P-body proteins CAR-1 and CGH-1 in oocytes. However, CCT does not appear to affect the condensation of MEX-3, the P-granule proteins PGL-1 and MEG-3, or the stress granule proteins PAB-1 and TIAR-2. Taken together, we conclude that the CCT chaperonin selectively modulates RNA-binding proteins in the germline. Our results suggest that improper folding by CCT of one or more substrates in the germline, leads to aggregation for a subset of RNA-binding proteins. Possible models to explain our results include CAR-1 and CGH-1 as substrates directly folded by CCT, or alternatively, an indirect regulator of RNA-binding proteins may be a CCT substrate. We are currently testing whether subunits of Prefoldin, a co-chaperone of CCT, have similar roles as CCT subunits on CAR-1 and CGH-1 condensation.

(2023) Development "An interview with Aimee Jaramillo-Lambert."

[Development, 2023]

Aimee Jaramillo-Lambert is currently an Assistant Professor at the University of Delaware. Aimee was awarded the Society for Developmental Biology 2023 Elizabeth D. Hay New Investigator Award in recognition of her outstanding research in developmental biology during the early stages of her independent career. We caught up with Aimee over a video call to talk about her research into sexual reproduction in Caenorhabditis elegans, the importance of mentorship, and what drives her work in diversity, equity and inclusion.

Watkins, Brooklynne et al. (2021) International Worm Meeting "Characterization of stress-induced phase transitions in the C. elegans germline"

Pestrue, Chloe, Sharp, Katherine, Schisa, Jennifer, Watkins, Brooklynne, Breton, Elizabeth, Elaswad, Mohamed

[International Worm Meeting, 2021]

Many RNAs and RNA-binding proteins undergo condensation to form ribonucleoprotein (RNP) granules in the germline. The triggers and regulation of condensate dynamics are not yet well understood; however, stresses such as extended meiotic arrest can affect condensate size. During our investigation of regulators of RNA-binding protein (RBP) condensation, we observed unexpected dispersal of PGL-1 protein in control experiments. Using the DAF-16::GFP stress reporter, we determined that our imaging methods were inadvertently inducing a stress response. When worms were imaged immediately after slide preparation, DAF-16 remained cytosolic; however, extended time on agarose pads induced nuclear translocation of DAF-16. Our preliminary results indicate this imaging stress induces condensation of MEX-3 and CGH-1, dispersal of PGL-1 and GLH-1, and has no effect on TIAR-2, PAB-1, and MEG-3. In arrested oocytes, this stress results in dispersal of PGL-1, but not of MEX-3 and CGH-1. As a start to characterize the imaging stress, a time course study revealed that DAF-16 trans-locates to nuclei within 20 minutes; therefore, we conclude researchers studying condensation of RBPs need to be cautious and rigorous in their imaging methods. We are currently testing additional stress reporters to further characterize the type of stress inducing phase transitions. We have also begun investigating the question of why RNA binding proteins in arrested oocytes respond differently to stress by characterizing their properties. Our preliminary results suggest the large PGL-1 granules in arrested oocytes, but not MEX-3 or CGH-1 granules, are sensitive to hexanediol, indicating hydrophobic interactions are required for PGL-1 granule integrity. In heat stress experiments, MEX-3 condensation increases in both arrested and non-arrested oocytes. However, PGL-1 becomes dispersed in non-arrested oocytes. We are also currently performing FRAP experiments to determine the mobility of these RBPs in large RNP granules. Taken together, we conclude that heat stress and our inadvertent imaging stress differentially affect the condensation of RNA-binding proteins in the germline. The differences among RBPs correlate with the degree of their liquid-like properties based on preliminary hexanediol data. Our results also suggest that the large RNP granules in arrested oocytes may contain multiple phases, similar to what has been observed for P granules in embryos.

Silverman PH (1999) Science "C. elegans as a model."

Silverman PH

[Science, 1999]

Elizabeth Pennisi, in her excellent commentary "Worming secrets from the C. elegans" (News Focus, 11 Dec 1998, p.1972), states that "The first person to sense that the worm might take on such a prominent role in biology was molecular biologist Sydney Brenner." I am sure that Brenner would wish to acknowledge the role that Ellsworth C. Dougherty played in this matter. Dougherty originally described in 1949, "[a] new species of the free-living nematode genus Rhabditis of interest in comparative physiology and genetics".

Vande Waa EA (1991) Parasitol Today "Chemotherapy of filariases."

Vande Waa EA

[Parasitol Today, 1991]

The filarial parasites that affect humans most seriously include Onchocerca volvulus, Wuchereria bancrofti, Brugia malayi and Loa loa. In general, these species cause disease that is debilitating long before it is fatal, producing clinical manifestations such as general malaise, pruritus and lichenification of the skin, lymphangitis, elephantiasis and blindness(1). As a result, infection with any one of these organisms is physically, as well as economically, devastating. Currently, the pharmacological armamentarium with which to treat filarial infections is very limited and many of the drugs that are known to be efficacious against these worms may produce side effects that cause extreme discomfort. Here, Elizabeth Vande Waa describes the quest for new chemotherapeutic approaches for the treatment of filarial infections.

Harris JB et al. (2020) Toxicology "Zebrafish CYP1A expression in transgenic Caenorhabditis elegans protects from exposures to benzo[a]pyrene and a complex polycyclic aromatic hydrocarbon mixture."

Dinyari A, Harris JB, Hartman JH, Meyer JN, Wilson JY, Luz AL

[Toxicology, 2020]

Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly-contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.

Macchietto, Marissa et al. (2017) International Worm Meeting "Hybrid assembly of the genome of the entomopathogenic nematode Steinernema carpocapsae."

Macchietto, Marissa, Mortazavi, Ali, Rodriguez, Bryan, Serra, Lorrayne

[International Worm Meeting, 2017]

Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most well-studied entomopathogens, due to its broad lethality to diverse insect species, and to its effective commercial use as a biological control agent for insect pests. For this reason, it has become an important genetic model for studying parasitism, pathogenesis, and symbiosis. We used a newly published hybrid assembly pipeline to assemble the best genome of S. carpocapsae to date, comprising 86,259,276 bp in 86 scaffolds, with an N50 of 4.03 Mb, from a combination of 75X coverage Pacbio and 130X coverage Illumina reads. We found that 90% of the genome is represented by the 22 largest scaffolds. RNA-seq data from 17 developmental stages spanning the embryo to adult stages were used to help predict 22,295 gene models, a major reduction in the number of genes from the previously published assembly by Dillman et al. 2015, which has 28,313 genes, and an increase in the number of genes relative to a high contiguity S. carpocapsae Breton strain assembly (N50=1.24), which has 16,333 genes. Using this new genome, we infer the potential chromosomal origins of our scaffolds by comparing them to C. elegans using shared one-to-one orthologs and find that many of the largest scaffolds correspond primarily to single chromosomes in C. elegans. We also investigate a potential large 1.2 Mb duplication in the genome, and delve into gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations will provide a good foundation for new comparative genomic and gene expression studies.