Nowakowski, Aidan et al. (2019) International Worm Meeting "Regulation of DNA repair pathway to ensure gamete quality."

Checchi, Paula, Rosenkranse, Erika, Nowakowski, Aidan, Andrews, Nicolas

[International Worm Meeting, 2019]

Meiotic recombination requires the formation and repair of genome-wide, programmed double-stranded breaks (DSBs). Repair of meiotic DSBs requires homologous recombination (HR), an error-free repair pathway required for crossover formation. Accordingly, error-prone pathways such as non-homologous end joining (NHEJ) are not favored during meiosis due to their propensity for generating mutations and their inability to form chiasmata. How error prone repair pathways are suppressed during meiosis is not well understood. Here, we demonstrate a role of for Mi2, the core ATPase subunit of the NuRD chromatin remodeling complex, in coordinating the repair of DSBs and maintaining genomic stability through multiple mechanisms. Our data reveal that the conserved Mi2 homologs CHD-3 and LET-418 promote HR, though in their absence, NHEJ is engaged as a secondary DNA repair mechanism to prevent persisting damage in gametes. In support of this, our findings indicate that a population of DSBs are repaired via NHEJ in situations with compromised LET-418 activity. Though HR mechanisms remain partially intact in let-418 mutants, their germ lines possess multiple defects including increased corpses and presence of structurally abnormal chromosomes in oocytes. These data are corroborated by molecular evidence wherein let-418 mutants have elevated expression of several NHEJ components, indicating a role for NuRD in the transcriptional regulation of repair genes during meiosis. Intriguing, our data also reveal that loss of LET-418 leads to upregulation of HR machinery, which we attribute to increased genomic stress in mitotically dividing germ cells, and we are currently investigating the causes and consequences of this. As these genes involved in these processes are highly conserved throughout eukaryotes, our findings have implications for understanding how Mi2 may contribute to the prevention of human disease states such as infertility and cancer.

Preston S et al. (2017) FASEB J "Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain."

Sternberg PW, Ansell BRE, Andrews KT, Nowell C, Chang BCH, Hofmann A, Crawford S, Korhonen PK, Baell J, Gijs MAM, Fisher GM, Young ND, Preston S, Mouchiroud L, Gasser RB, Jabbar A, Auwerx J, Davis RA, McGee SL, Cornaglia M

[FASEB J, 2017]

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Wang S et al. (2020) Plant Physiol Biochem "Composition of peony petal fatty acids and flavonoids and their effect on Caenorhabditis elegans lifespan."

Zheng S, Xue J, Wang S, Zhang S, Zhang X, Xu D, Xue Y

[Plant Physiol Biochem, 2020]

The colorful petals of tree peony (Paeonia suffruticosa Andrews) are widely used as a source of additives in food, fragrances, and cosmetics. However, the nutritional composition of peony petals is undetermined, thereby limiting utility and product development. In this work, fresh petals of 15 traditional Chinese tree peony cultivars were selected to analyze the composition of soluble sugars, starch, and soluble protein. Extracted fatty acids (FAs) and flavonoids from petals were characterized by GC-MS and UPLC-triple-TOF-MS, respectively. The oxidative stress resistance (generated by paraquat) effects of petal extracts of three cultivars were also investigated in the model organism Caenorhabditis elegans. Our results showed that the petals were highly enriched in soluble sugars. 11 FAs were found in tree peony petals, and their compositions were similar to that of tree peony seeds. A total of 56 flavonoids were detected in tree peony petals, 28 of which were reported for the first time in tree peony petals, indicating that UPLC-triple-TOF-MS can improve the identification efficiency of flavonoids. Further analysis of tree peony petal metabolites indicated that anthocyanidin and flavonol composition might be used as specific chemotaxonomic biomarkers for cultivar classification. Flavonoids, linoleic acid, and -linolenic acid (ALA) in petals might provide antioxidant activity. 150mg/L of petal extracts of all three tested cultivars increased the lifespan of C. elegans. It was suggested that the petal extracts possessed anti-aging effects and oxidative stress resistance. These results highlight that tree peony petals can serve as natural antioxidant food resources in the future.

Fassnacht, N. et al. (2019) International Worm Meeting "Chromatin remodeling mediates repair of DNA damage in C. elegans germ cells."

Peruffo , A., Fassnacht, N., Checchi, P.

[International Worm Meeting, 2019]

Chromatin remodeling proteins are required for multiple aspects of DNA repair. This is particularly important in germ cells, wherein defective repair affects gamete quality. This in turn leads to infertility, miscarriage, and chromosomal disorders. Previously, we discovered a role for the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex in the repair of programmed DNA lesions. Loss of the catalytic component of NuRD, known as the Mi2 complex, disrupts repair of meiotic DSBs1. Here we investigated the role of the Mi2 component LET-418 in response to several forms of exogenous DNA damage. We assessed embryonic viability in response to several mutagens including nitrogen mustard and cisplatin, which cause inter- and/or intra-strand crosslinks (ICLs), a byproduct of replication errors. Our data reveal reduced embryonic viability in a let-418 loss-of-function mutant when mitotically dividing germ cells are exposed to these agents. This is consistent with previous findings that Mi2 has a role in the repair of double-stranded breaks, which can be formed from ICLs. We are currently assessing the molecular nature as to how LET-418 responds to ICLs and other types of DNA lesions, such as those caused by hydroxyurea and UVC. Overall, our findings support a role of Mi2 in maintaining genomic stability in multiple contexts. 1Turcotte, C.A., Sloat, S.A., Rigothi, J.A, Rosenkranse, E., Northrup, A., Andrews, N.P., and Checchi, P.M. (2018). Maintenance of genome integrity by Mi2 homologs CHD-3 and LET-418 in Caenorhabditis elegans. Genetics. 208:1-17.

Todd R Heallen et al. (2005) International Worm Meeting "An RNAi-based suppressor screen for components of the Aurora B kinase pathway"

Jill M Schumacher, Todd R Heallen

[International Worm Meeting, 2005]

Proper execution of mitosis requires accurate segregation of newly replicated DNA into each of two newly formed cells. Genetic instability can be caused not only by defects in the segregation process itself, but also by failures in cytokinesis that can lead to the formation of polyploid cells. Our studies focus on a highly conserved family of mitotic regulators, the Aurora kinases. These proteins comprise a class of serine/threonine kinases that are essential for mitotic events such as centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis (Andrews et. al., 2003). AIR-2, the C. elegans homolog of Aurora B, has been shown to interact with a variety of substrates to facilitate processes such as chromosome segregation and cytokinesis. Depletion of AIR-2 via RNA-mediated interference (RNAi) leads to embryonic lethality marked by the accumulation of polyploid one-cell embryos (Schumacher et. al., 1998). To understand the multiple functions of the AIR-2 kinase and its mode of regulation, additional components of the AIR-2 regulatory pathway need to be identified. To identify these components, we conducted a genome-wide RNAi-based screen to identify suppressors of a temperature-sensitive hypomorphic air-2 mutant, air-2 (or207ts). air-2 (or207ts) harbors a missense mutation in the predicted kinase domain and results in embryonic lethality and characteristic unicellular polyploid embryos reminiscent of air-2 (RNAi) when incubated at the restrictive temperature (25C) (Severson et. al., 2000). We identified air-2 (or207ts) suppressors by screening for RNAi constructs that resulted in the production of viable progeny at restrictive temperatures. Thus far, our screening efforts have uncovered 56 suppressor candidates whose depletion via RNAi conferred weak suppression (<0.5% increase in the production of hatched embryos) of air-2 (or207ts) ts lethality at 25C. However, upon retesting ten candidates at a semi-permissive temperature (20C), two exhibited stronger suppression (a 2.5% increase in hatched embryos over controls). We are currently testing the remaining candidates for suppression of the air-2(or207ts) lethality at semi-restrictive temperatures. Further characterization of selected candidates will be presented.

Grundahl K et al. (1997) International C. elegans Meeting "unc-41 ENCODES A PRESYNAPTIC PROTEIN"

Rand JB, Grundahl K, Eustance RJ, Crowell T

[International C. elegans Meeting, 1997]

unc-41 mutants display a coiling, uncoordinated behavior, resistance to inhibitors of cholinesterase, elevated levels of acetylcholine, slow growth, and small adult size. This set of phenotypes is associated with defects in acetylcholine release and/or synaptic vesicle function. In addition, unc-41 animals display a defecation-expulsion defect associated with defective GABA function. It is therefore likely that unc-41 encodes a protein important for the release of most or all neurotransmitters. We cloned the gene by transposon tagging, and we have isolated and sequenced unc-41 genomic and cDNA clones. Northern and cDNA analysis reveals that there are two unc-41 transcripts. These are 5.3 and 4.5 kb long; they are both rare and appear to be transcribed from different promoters. The structural difference between the two transcripts arises from the presence of additional exons upstream of the 4.5 kb transcript and its presumed promoter. The gene encodes a pair of nested hydrophilic proteins (1650- and 1400-amino acids). These have slight similarity to the AP50 clathrin adaptor protein family (which functions in membrane trafficking and endocytosis) and a strong similarity to the 1260-amino acid stonedB protein of Drosophila (Andrews et al., 1996). The stoned locus is a complex locus which encodes two structurally unrelated proteins. There are genetic interactions between the stoned and shibire loci (shibire encodes the endocytosis protein dynamin); it is therefore hypothesized that the stoned protein(s) also function in endocytosis. The sequence similarity is limited to the 500-amino acid C-terminal domain of the UNC-41 and stonedB proteins. Immunolocalization studiesindicate that the UNC-41 proteins are located at synapses in most or all neurons, and that they are not associated with synaptic vesicles. We have analyzed a large collection of unc-41 alleles - this includes 27 alleles isolated in our lab, 9 MRC alleles, and two MIT alleles. Many of them have residual immunostaining, and some alleles have only partial behavioral impairment. (Supported by a grant from NINDS.)

Han, X. et al. (2011) International Worm Meeting "Mechanisms Involved in Regulating the Activity and Localization of a Microtubule-depolymerizing Kinesin in the One-cell Stage C. elegans Embryo."

Cheung, K., Srayko, M., Han, X.

[International Worm Meeting, 2011]

Microtubules are required for multiple cellular processes including mitosis, cytokinesis, and vesicle transportation. Factors that regulate microtubule dynamics in the cell help determine the final form and precise cellular role of many cytoskeletal structures. Among the known modulators of microtubule dynamics, we are specifically interested in the microtubule-depolymerizing kinesins of the kinesin-13 family, such as KLP-7 (CeMCAK). In C. elegans, KLP-7 locates to the kinetochore and the centrosome. It has been implicated in regulating microtubule outgrowth at the centrosome because loss of KLP-7 results in an increase in the number of centrosomal microtubules (Srayko et al., 2005, Schlaitz et al., 2007). Extensive work from other labs on the vertebrate homologues of KLP-7 indicates that they are negatively regulated through phosphorylation by the Aurora kinases (Andrews et al., 2004, Lan et al., 2004, Ohi et al., 2004, Schlaitz et al., 2007, Zhang et al., 2007 and 2008). In order to understand how KLP-7 is regulated in the cell, we tested the possibility that Aurora kinases are directly involved. 2D gel-electrophoresis revealed an alteration in the ratio of potential KLP-7 phosphorylation variants in lysates from either air-1(RNAi) (Aurora A-depleted) or air-2(RNAi) (Aurora B-depleted) embryos, compared to wild type. Furthermore, we found that both AIR-1 and AIR-2 kinases phosphorylate KLP-7 in vitro. We used a combination of mass spectrometry, similarity to data on the vertebrate homologues, and an Aurora kinase phospho-site prediction algorithm to identify potential in vivo phosphorylation sites within KLP-7 (Zhou et al., 2004). We are currently performing a structure-function analysis to determine which putative Aurora sites are required for KLP-7's intracellular location and/or its depolymerase activity at the centrosome. Results obtained thus far indicate that mutating one of the C-terminal Aurora sites from serine to glutamic acid (to mimic constitutive phosphorylation) or to alanine (to mimic non-phosphorylation) interferes with KLP-7 function but not its ability to target to centrosomes or kinetochores.

Todd Heallen et al. (2006) Development & Evolution Meeting "Novel Regulation of the Aurora B Kinase Pathway by a Conserved Member of the AAA-ATPase Protein Family"

Todd Heallen, Jill Schumacher

[Development & Evolution Meeting, 2006]

Timely and error-free progression of the cell cycle is fundamental to the development and proper function of eukaryotic organisms. As such, eukaryotes have evolved complex regulatory mechanisms to facilitate development and cell maintenance. Our studies are focused on an evolutionarily conserved family of mitotic proteins called the Aurora kinases. These proteins comprise a class of serine/threonine kinases that are essential for mitotic events such as centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis (Andrews et. al., 2003). AIR-2, the C. elegans homolog of Aurora B, has been shown to interact with a variety of substrates to facilitate processes such as chromosome segregation and cytokinesis. Depletion of AIR-2 via RNA-mediated interference (RNAi) leads to embryonic lethality at the one cell stage (Schumacher et. al., 1998). A temperature-sensitive (ts) mutation, air-2 (or207ts) confers a similar phenotype at restrictive temperatures (Severson et. al., 2000). Our current goal is to elucidate the AIR-2 regulatory pathway by identifying the components working in concert with AIR-2. We therefore conducted a genome-wide RNAi screen to identify suppressors of air-2(or207ts) lethality. We have screened through an RNAi construct library comprising nearly every gene in the C. elegans genome (approximately 17,000 genes). The strongest RNAi suppressor identified from this screen encodes a putative C. elegans AAA-ATPase and confers ~72.6% viability to the air-2(or207ts) strain at 20&ordm;C as compared with 1% viability for air-2(or207ts) alone. For simplicity purposes, we have tentatively named this suppressor abs-1 (Aurora B Suppressor-1). To understand the relationship between air-2 and abs-1, functional analyses are being performed. In vitro binding and kinase assays reveal that ABS-1 can bind directly to AIR-2 and inhibit its kinase activity. In addition, Western analysis revealed that AIR-2 levels are stabilized in abs-1(RNAi) embryo extracts as compared to controls. Furthermore, immunostaining studies reveal that AIR-2 localization is disrupted in abs-1(RNAi) embryos. Hence, our data suggests that abs-1(RNAi) can suppress the air-2(or207ts) lethality by stabilizing AIR-2 levels and by controlling the localization and kinase activity of AIR-2. We are therefore examining the possibility that the AIR-2 complex is regulated by ABS-1 via proteolytic and non-proteolytic pathways.

Todd Heallen et al. (2007) International Worm Meeting "Novel Regulation of the Aurora B Kinase Pathway by a Conserved Member of the AAA-ATPase protein family."

Jill Schumacher, Todd Heallen

[International Worm Meeting, 2007]

Timely and error-free progression of the cell cycle is fundamental to the development and proper function of eukaryotic organisms. As such, eukaryotes have evolved complex regulatory mechanisms to facilitate development and cell maintenance. Our studies focus on an evolutionarily conserved family of mitotic proteins called the Aurora kinases. These proteins comprise a class of serine/threonine kinases that are essential for mitotic events such as centrosome maturation, spindle assembly, chromosome segregation, and for cytokinesis (Andrews et. al., 2003). AIR-2, the C. elegans homolog of Aurora B, has been shown to interact with a variety of substrates to facilitate these processes. AIR-2 depletion via RNA-mediated interference (RNAi) leads to embryonic lethality at the one cell stage (Schumacher et. al., 1998). A temperature-sensitive (ts) mutation, air-2 (or207ts) confers a similar phenotype at restrictive temperatures (Severson et. al., 2000). My current goal is to elucidate the AIR-2 regulatory pathway by identifying the components that work in concert with the AIR-2 kinase. To this end, I conducted a genome-wide RNAi screen to identify suppressors of air-2 (or207ts) lethality. One of these suppressors, which we have named cdc-48.3, encodes a C. elegans homolog of the molecular chaperone Cdc48/p97. Yeast and vertebrate homologs of Cdc48 associate with subsets of cofactors to mediate spindle disassembly, proteolysis, and recruitment of proteins to the mitotic spindle. (Wang et. al., 2003) (Cheeseman and Desai., 2004) Altogether, the above findings suggest that Cdc48 and its cofactors regulate the chromosomal passengers, and that this regulation may be evolutionarily conserved. To understand the relationship between air-2 and cdc-48.3, a number of functional analyses have been performed. Western analysis revealed that AIR-2 levels are stabilized in cdc-48.3 (RNAi) embryo extracts as compared to controls. Quantitative immunostaining studies showed that this stabilization occurs primarily at mitotic exit. Interestingly, in vitro binding and kinase assays revealed that CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity. To test the effect of CDC-48.3 on AIR-2 kinase activity in vivo, I examined the levels of phosphorylated ICP-1 (pICP-1), a substrate of the AIR-2 kinase, and autophosphorylated AIR-2 (pAIR-2) in cdc-48.3 (RNAi) embryos. These data revealed that phosphorylated ICP-1 and pAIR-2 levels are upregulated in cdc-48.3 (RNAi) embryos. Altogether, these data suggest that CDC-48.3 directly interacts with the AIR-2 kinase, resulting in the inhibition of AIR-2 kinase activity and AIR-2 degradation at mitotic exit. .