Vader G et al. (2014) Dev Cell "HORMA domains at the heart of meiotic chromosome dynamics."

Musacchio A, Vader G

[Dev Cell, 2014]

HORMA domain proteins are required for the careful orchestration of chromosomal organization during meiosis. Kim et al. (2014) and Silva et al. (2014) now provide structural and functional insights into the roles of C. elegans HORMA proteins, revealing parallels to the function of the HORMA protein MAD2 in mitotic checkpoint signaling.

Davo Martinez, Carlota et al. (2021) International Worm Meeting "Persistent DNA repair complex binding in the absence of DNA damage excision impairs neuron functionality"

Ribeiro-Silva, Cristina, Muniesa Vargas, Alba, Theil, Arjan, Davo Martinez, Carlota, Vermeulen, Wim, Lans, Hannes

[International Worm Meeting, 2021]

Nucleotide excision repair (NER) is a major DNA repair pathway that removes a large variety of helix-distorting DNA damage. Hereditary mutations in NER genes that encode proteins that make up the core NER machinery, such in endonucleases XPF and XPG, can give rise to the cancer prone disorder xeroderma pigmentosum (XP) or to more severe disorders in which XP is combined with progressive neurodegeneration or progeroid Cockayne syndrome (CS) features, called XP-CS. Intriguingly, mutations in other factors of the core NER complex give rise to a milder phenotype. It is not properly understood why mutations in genes that act in the same DNA repair pathway cause different types of disease. Here, we use C. elegans as a model to study the phenotypic impact of different mutations in the evolutionary conserved NER pathway. We found that XPF-1 or XPG-1 deletion mutants show severe developmental arrest and neuronal dysfunction upon induction of UV damage. In the absence of XPF-1 or XPG-1, core NER-intermediates accumulate at DNA damage and stay stably bound to DNA. We hypothesized that this could shield the lesion from other DNA repair pathways and block transcription, which could cause this severe phenotype. Intriguingly, we found that inhibiting the binding of the core NER-intermediates to DNA in XPF-1 or XPG-1 worms alleviated the severe phenotype, confirming that the accumulation of repair intermediates can indeed be more toxic for cells than the damage itself. Together, these results strongly suggest that the persistence of NER intermediates adversely affects cell functionality, which may be a plausible explanation for why mutations in XPF or XPG can lead to more severe disease features than mutations in other core subunits.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Kazatskaya, Anna et al. (2020) MicroPubl Biol

de Bono, Mario, Amin-Wetzel, Niko, Sengupta, Piali, Philbrook, Alison, Kazatskaya, Anna, Yuan, Lisa

[MicroPubl Biol, 2020]

A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.

Mendel JE et al. (1991) International C. elegans Meeting "The Expresson of G protein alpha Genes in C. elegans"

Lochrie MA, Simon MI, Kim UJ, Sternberg PW, Mendel JE

[International C. elegans Meeting, 1991]

G proteins are a class of guanine nucleotide binding proteins that act in transmembrane signal transduction by physically coupling cell surface receptors to effector proteins. They comprise three subunits: alpha, , and gamma. The a subunit undergoes a guanine nucleotide exchange and hydrolysis cycle and, in most cells, interacts with effector molecules. G protein a subunits are members of a large family of proteins. For example, 16 G a subunit genes have been cloned from the mouse. Using PCR-based methodology, we have cloned five G a subunit genes from C. elegans. Two of them, goa-l and gqa-l are homologous to mammalian and Drosophila Galpha(o) and Galpha(q) respectively. The remaining three, gpa-l, gpa-2, and gpa-3, appear to be novel to C. elegans. Fino Silva and Plasterck [JMB 215, 483 (1990)], have also described the gpa-2 gene. We have fused the 5' flanking regions of goa-l and gpa-l to the E. coli lacZ gene using Andy Fire's modular vectors [Gene 93, 189 (1990)]. The staining pattern for the goa-l-lacZ construct indicates that goa-l is expressed in a large number of neurons, both in the circumpharyngeal nerve ring and the ventral cord. This apparent expression pattern is consistent with the observation that GaO is abundant in neural tissue in other organisms. The staining pattern for the gpa-l-lacZ construct indicates that gpa-l is expressed in a small number of cells in the pharynx, four cells in the circumpharyngeal nerve ring, and what appears to be one of the phasmid neurons. In males, staining is also seen in at least one of the spicule neurons.

Rooney, John P et al. (2013) International Worm Meeting "Developmental Exposure to Ultraviolet C Radiation Results in Altered Energy Production Later in Life in Caenorhabditis elegans."

Ji, Alex, Leung, Maxwell, Rooney, John P, Bodhicharla, Rakesh, Ryde, Ian, Bess, Amanda, Meyer, Joel

[International Worm Meeting, 2013]

Mitochondrial genomes encode for 13 proteins that are essential components of the electron transport chain (ETC) and are normally present at between 1000 and 100,000 copies per cell. However, this number is greatly reduced during specific developmental stages, representing a potential critical window for mitochondrial genotoxicant exposure. Mitochondrial DNA (mtDNA) is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including absence of Nucleotide Excision Repair (NER). NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and many environmental toxicants. In the current experiment we tested the hypothesis that UVC induced mtDNA damage during larval development would alter energy production throughout the life of the nematode. ATP levels were measured as luminescence using the firefly luciferase expressing PE255 glp-4(bn2) strain (generously provided by Cristina Lagido, University of Aberdeen), and both mtDNA and nuclear DNA copy numbers were measured via quantitative real time PCR. We exposed first larval stage PE255 nematodes to a serial UVC dose that results in the accumulation of mtDNA damage while allowing for repair of nuclear DNA damage, and measured ATP levels, mtDNA and nucDNA copy numbers every 48 hours for 10 days. Immediately after treatment, ATP levels were nearly equal, however at post treatment days 2 through 10 ATP levels were 50-70% lower in UVC treated nematodes. Interestingly, there was no detectable change in either mtDNA or nucDNA copy numbers throughout the time course. These results indicate that mtDNA damage during larval development can alter energy production throughout the life of the nematode, and highlights the potential for a critical window of exposure to mtDNA damage. Furthermore, while it is well established that genetic manipulation of expression of ETC components results in reduced ATP levels and lifespan extension, our data suggest the same phenotypes can be induced via environmental exposures.

Hagy, Kevin et al. (2017) International Worm Meeting "Do defects specific to crossover recombination inhibit the DNA damage response in C. elegans?"

Bhalla, Needhi, Giacopazzi, Stefani, Conrad, Barbara, Hagy, Kevin

[International Worm Meeting, 2017]

Diversity in the animal kingdom is established through mutations, gene flow, and sexual reproduction. During gamete production, genetic material is exchanged between maternal and paternal chromosomes via a process called crossover recombination. In C. elegans, recombination between homologs is essential for proper segregation during anaphase I. The steps of recombination occur as follows: 1. SPO-11 introduces DNA double-strand breaks (1), 2. RAD-51-dependent single-end strand invasion favors repair from homologous chromosomes (4,5), and 3. crossover(CO) -specific DNA repair is promoted by MSH-4, MSH-5 and ZHP-3 (2,3). Thus, loss of CO promoting factors, such as MSH-4, MSH-5 and ZHP-3, should lead to persistent DNA damage and an increase in activation of DNA damage induced apoptosis, a process dependent on the conserved apoptosis activator EGL-1. However, Silva et al showed that loss of these CO-promoting factors fails to activate DNA damage induced apoptosis. Further, these factors appear to be required for initiation of the DNA-damage checkpoint since apoptosis decreases in msh-4, msh-5, and zhp-3 mutants after IR radiation (6). We present an alternate explanation of these results: When we mutate a conserved upstream regulatory region of egl-1, msh-5 and zhp-3 mutants now exhibit an increase in apoptosis and this increase is dependent on the DNA damage checkpoint protein HUS-1. We hypothesize that when CO recombination is specifically disrupted, DNA damage induced apoptosis is inhibited, potentially to promote repair over removal. We plan to 1) identify the regulatory factor(s) that bind EGL-1's regulatory region to limit apoptosis when CO recombination is defective and 2) test whether this inhibition of apoptosis is mechanistically linked to characterized meiotic feedback mechanisms that are activated by defects in recombination.

Nusser, Stefanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The LIN-12/NOTCH target ttr-11 promotes secondary vulval cell fate specification"

Hajnal, Alex, Rimann, Ivo, Farooqui, Sarfarazhussain, Nusser, Stefanie

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The vulva of the hermaphrodite is an excellent model organ to study cell fate specification by intercellular signals. The combined action of the strongly conserved RTK/RAS/MAPK, NOTCH and WNT signaling pathways determines the primary (1) and secondary (2) fate. So far, only genes mediating the inhibitory function of LIN-12/NOTCH have been described, while genes required to instruct the 2 fate remain unknown. We have identified in an RNAi screen ttr-11 as a NOTCH target gene required for 2 cell fate specification. TTR-11 belongs to a nematode-specific protein family called the transthyretin-like family that shows weak similarity to mammalian transthyretin (TTR) proteins. Human TTR is a plasma protein required to transport thyroid hormone and vitamin A. Interestingly, mutations in human TTR cause transthyretin amyloidosis characterized by the formation of fibrillar TTR aggregates in peripheral tissues (Bergstrom, Gustavsson et al. 2005). Moreover, TTR has been proposed to affect the processing of the amyloid precursor protein (APP) (Costa, Ferreifa-da-Silva et al.2008). By investigating the expression pattern of ttr-11 in lin-12 gain- and loss-of-function mutants, we found that LIN-12/NOTCH signaling regulates ttr-11 expression in 2 VPCs. The ttr-11 ( zh102 ) deletion allele partially suppresses the vulvaless phenotype of the weak lin-12 ( n302 ) gain-of-function allele. Moreover, in ttr-11 ( zh102 ); lin-15 ( n309 ) double mutants, more VPCs adopt the 1 cell fate than in lin-15 ( n309 ) single mutants, suggesting that TTR-11 promotes 2 fate specification. We are currently performing a functional characterization of TTR-11 to identify its binding partners and characterize its mechanism of action in the LIN-12/NOTCH pathway. Our research contributes to a better understanding of the signaling cells cross talk in the VPCs.

O'Hagan, Robert et al. (2019) International Worm Meeting "The Tubulin Code: Writers, erasers, and readers specialize cilia."

Zhang, Winnie, Bellotti, Sebastian, Smith, Harold, De Stasio, Elizabeth, Nguyen, Ken, Gu, Amanda, O'Hagan, Robert, Morash, Maggie, Hall, David, Power, Katlin, Golden, Andrew, Barr, Maureen

[International Worm Meeting, 2019]

In eukaryotic cells, microtubules (MTs) act as highways, and molecular motors, such as kinesins, are cargo trucks. The "Tubulin Code" posits that tubulin isotypes and tubulin post-translational modifications such as polyglutamylation (addition of glutamate side-chains) act like signposts that regulate the activity of motors and the structure of MTs (Verhey and Gaertig, 2007). Cilia from all eukaryotes share conserved 9+0 or 9+2 MT ultrastructures and well-characterized molecular motors, and are therefore a powerful model with which to discover the mechanisms by which the Tubulin Code acts. Cephalic male (CEM) cilia possess a unique architecture: 9 MT doublets splay into 18 singlets, which is essential for motor-based transport and the release of ciliary extracellular vesicles. We showed that in CEM cilia, the a-tubulin TBA-6 and the glutamylase TTLL-11 write the Tubulin Code, and the deglutamylase CCPP-1 acts as an eraser, while the kinesin-3 KLP-6 and the kinesin-2 OSM-3 are readers of the Tubulin Code [Morsci and Barr, 2011; O'Hagan et al, 2011; Silva et al, 2017; O'Hagan et al, 2017]. In amphid channel cilia, the Tubulin Code performs a different function. In ccpp-1 amphid channel cilia, hyperglutamylation produces ciliary degeneration as detected by dye-filling and transmission electron microscopy. In humans and mice, mutations in Ccp1, the homolog of C. elegans ccpp-1, result in neurodegeneration. To discover molecules involved ciliary MT stability, we took candidate and forward genetic screening approaches to find modifiers of the ccpp-1 Dyf phenotype. We find that the ccpp-1 ciliary degeneration phenotype was suppressed by mutation in any of three glutamylases ttll-4, ttll-5, and ttll-11 or the never in mitosis kinase nekl-4 (see poster by K. Power). NEKs have been implicated in ciliary length regulation and human ciliopathies. This work should uncover novel pathways that establish, maintain, and modify MT architecture and motor function.

Niacaris TR et al. (1998) Worm Breeder's Gazette "Expression patterns of candidate serotonin receptors"

Niacaris TR, Avery L

[Worm Breeder's Gazette, 1998]

Serotonin increases the rate of pharyngeal pumping and egg laying, decreases foraging behavior and is required for proper male mating behavior. Serotonin can influence pharyngeal pumping rate by stimulating MC, a motor neuron which stimulates pharyngeal muscle contraction, or by acting directly on pharyngeal muscle. To determine the receptor or receptors that mediate this response, we have searched the genomic sequence for candidate genes having sequence identity to known vertebrate and invertebrate serotonin receptors. We found seven candidate genes using this method: C02D4.2, C09B7.1, C52B11.3, F01E11.5, F14D12.6, F59C12.2 and M03F4.3. Two of these genes (F59C12.2 and M03F4.3) were previously identified as possible serotonin receptor candidates (Ribeiro and Hamdan IWM97, p278.) We were not able to distinguish octopamine receptors from metabotropic serotonin receptors based on sequence. In addition, there are no sequences in the C. elegans database with significant similarity to the ionotropic serotonin receptor subclass, 5-HT3. We have created GFP fusion constructs of four of the candidate genes (C02D4.2, F01E11.5, F14D12.6 and F59C12.2) using overlap extension PCR. We directly injected these PCR products into worms to determine GFP expression patterns. F01E11.5 and F14D12.6 show limited expression outside the pharynx. The F01E11.5 construct expresses in a few unidentified neurons of the head, while F14D12.6 expression is most pronounced in the spermatheca of the adult. Two of the genes, C02D4.2 and F59C12.2, show pharyngeal expression of the fusion construct. F59C12.2 shows nearly ubiquitous pharyngeal and extrapharyngeal staining. C02D4.2 pharyngeal muscle expression is specific for the metastomal flap muscles, pm1, and the anterior terminal bulb muscles, pm6. The C02D4.2 construct also expresses in muscles of the head, the pharyngeal serotonergic neuron NSM, the ventral nerve cord and unidentified neurons in the nerve ring, head and tail regions. In males, C02D4.2 expresses in dorsal and ventral body muscles of the tail region. The construct also expresses in the male-specific diagonal muscles and serotonergic CP neurons. The expression pattern of C02D4.2 appears to be consistent with several known effects of serotonin. We are currently working to knock out both C02D4.2 and F59C12.2 using PCR detection of EMS-induced deletions of these genes.