Silva IF et al. (1990) J Mol Biol "Characterization of a G-protein alpha-subunit gene from the nematode Caenorhabditis elegans."

Plasterk RHA, Silva IF

[J Mol Biol, 1990]

A gene encoding the alpha-subunit of a guanine nucleotide binding regulatory protein (G-protein) was isolated from a library of genomic Caenorhabditis elegans DNA. The predicted coding region is colinear to related genes from mammals and the 356 amino acid residues show 63% sequence identity to e.g. rat Gi alpha 2. Three of the eight introns within the coding sequence are at exactly the same positions as those in a Drosophila G-protein alpha-subunit gene, and two of these are also conserved in the mammalian homologues. The nematode gene does not encode the cysteine residue that forms the substrate site for pertussis toxin-catalyzed ADP-ribosylation in several G-proteins. In spite of the similarity to mammalian G-protein alpha-subunit genes the gene can not unambiguously be categorized in one of the classes of G-proteins recognized in mammals (G alpha i, o, z, etc.). The position of the gene on the physical map of the animal was determined (chromosome V). The cloning and sequencing of this gene can be the starting point of reverse genetics experiments aimed at the isolation of animals mutated in a G-protein alpha-subunit gene.

Andre Pires da Silva (2005) WormBook "Pristionchus pacificus genetic protocols."

Andre Pires da Silva

[WormBook, 2005]

Silva M et al. (2014) Elife "Tomography gives a new dimension to an ancient organelle."

Silva M, Barr MM

[Elife, 2014]

Advances in sample preparation and electron microscopy have allowed the structure of cilia to be explored at an unprecedented level of detail.

Silva JP et al. (2010) Adv Exp Med Biol "The latrophilins, "split-personality" receptors."

Ushkaryov YA, Silva JP

[Adv Exp Med Biol, 2010]

Latrophilin, a neuronal "adhesion-G protein-coupled receptor", is the major brain receptor for alpha-latrotoxin, a black widow spidertoxin which stimulates strong neuronal exocytosis in vertebrates. Latrophilin has an unusual structure consisting of two fragments that are produced by the proteolytic cleavage of the parental molecule and that behave independently in the plasma membrane. On binding an agonist, the fragments reassociate and send an intracellular signal. This signal, transduced by a heterotrimeric G protein, causes release of calcium from intracellular stores and massive release of neurotransmitters. Latrophilin represents a phylogenetically conserved family of receptors, with orthologues found in all animals and up to three homologues present in most chordate species. From mammalian homologues, latrophilins 1 and 3 are expressed in neurons, while latrophilin 2 is ubiquitous. Latrophilin 1 may control synapse maturation and exocytosis, whereas latrophilin 2 may be involved in breast cancer. Latrophilins may play different roles during development and in adult animals: thus, LAT-1 determines cell fate in early embryogenesis in Caenorhabditis elegans and controls neurotransmitter release in adult nematodes. This diversity suggests that the functions of latrophilins may be determined by their interactions with respective ligands. The finding of the ligand of latrophilin 1, the large postsynaptic protein lasso, is the first step in the quest for the physiological functions of latrophilins.

da Silva AF et al. (2022) Neurotoxicology "JM-20 affects GABA neurotransmission in Caenorhabditis elegans."

Arantes LP, Soares MV, Nunez-Figueredo Y, Cordeiro LM, da Silveira TL, Soares FAA, Baptista FBO, Machado ML, Zamberlan DC, da Silva AF, Rodriguez EO

[Neurotoxicology, 2022]

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodiazepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a promising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the development of worms after 48h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM-20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, computational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model.

Silva, Malan et al. (2011) International Worm Meeting "Doublets' well kept secret: the sister singlets."

O'Hagan, Robert, Silva, Malan, Barr, Maureen, Nguyen, Ken, Hall, David H.

[International Worm Meeting, 2011]

Cilia are cellular antennae; in humans, defects in ciliary form or function give rise to ciliopathies. C. elegans sensory organs, or sensilla, consist of morphologically and functionally diverse arrays of cilia that mediate sensation. The length and shape of cilia depends on axonemal microtubule (MT) architecture. In the amphid channel cilia, the ciliary axoneme is anchored to the membrane by a region known as the transition zone (TZ). In the TZ, MT doublets are made of a 13 protofilament A-tubule and an attached 11 protofilament B-tubule. MT doublets are anchored to the membrane with "Y" links of unknown composition. Beyond the TZ, amphid channel cilia display two distinct zones classified using MT architecture. The middle segment consists of nine MT doublets that lack 'Y' links. The distal segment is composed entirely of MT singlets. Not all C. elegans cilia share this axonemal structure. For example, we have shown that cilia of the male cephalic neurons (CEMs) are composed of singlet MTs (1). We used high pressure freeze fixation and electron tomography to observe CEM cilia ultrastructure. By comparing cephalic and amphid channel cilia, we hope to understand at the molecular level how axonemes are specialized in form and function. We focused on transition from MT doublets to singlets and traced individual A- and B-tubules at the doublet-to-singlet transition. We found that the A- and B-tubules in middle segment doublets in CEM cilia separate into two sister singlets in the distal segment. These sister singlets are continuous to the distal segment of the cilium and spatially diverge from each other. Additionally we found that as compared to TZs of amphid neurons, CEM TZs span a longer distance. We are interested in determining how this spatial and structural disposition of the CEM MTs occurs, and how it affects ciliary motors. We are currently testing the hypothesis that tubulin post-translational modifications contribute to MT arrangement and stability in C. elegans sensory cilia (see Abstract by O'Hagan et al). 1. A. R. Jauregui, K. C. Q. Nguyen, D. H. Hall, M. M. Barr, The JCB 180, 973-988 (2008).

Andre Pires da Silva et al. (2003) International Worm Meeting "Evolution of sex determination mechanisms in nematodes"

Ralf J Sommer, Andre Pires da Silva

[International Worm Meeting, 2003]

While developmental control genes are highly conserved throughout the animal kingdom, sex determination genes and pathways evolve rapidly. In the genus Caenorhabditis, fast sequence evolution of signaling components involved in sex determination was observed. How then, do complex sex determination pathways arise during evolution? We are using a genetic approach to study sex determination in Pristionchus pacificus, a hermaphroditic nematode with a XX/X0 sex determination system (Sommer et al., 1996). In previous studies, this nematode has been shown to be distinct of C. elegans in many cellular and molecular mechanisms for vulva development. In a screen of 7000 gametes we isolated 8 sexually reversed mutants. Using X-chromosome-linked markers, we show that these animals are pseudomales with a XX karyotype. The hermaphrodite to male transformation is almost complete in the soma (development of spicules, rays and bursa in the tail and an one-armed gonad), but incomplete in the germline. Four transformers map to P. pacificus linkage group III, which is in large part syntenic to C. elegans chromosome III (Lee et al, submitted). We cloned Ppa-tra-1, the ortholog of the terminal sex determination gene of C. elegans, which is located on chromosome III. Two of the alleles are mutated in the conserved zinc-finger domain (66% identity compared to C. elegans). Regions outside of this domain, which are conserved in the Caenorhabditis genus and are involved in interacting with TRA-2, are not recognizable in P. pacificus. These results provide the first indication that sex determination genes upstream of the DM-domain proteins, such as mab-3, are conserved over large evolutionary distances. Furthermore, they suggest that most of the tra-1 specificity is given by the zinc finger domain or that there is a co-evolution of interacting partners outside this region. Heat-shock constructs with different domains of Ppa-tra-1 are being created to address this question. Sommer, R. J., Carta, L. K., Kim, S. Y. and Sternberg, P. W. (1996). Morphological, genetic and molecular description of Pristionchus pacificus sp n (Nematoda: Neodiplogastridae). Fundamental and Applied Nematology 19, 511-521.

da Silva TC et al. (2022) Neurochem Res "Exogenous Adenosine Modulates Behaviors and Stress Response in Caenorhabditis elegans."

Dalla Corte CL, Soares FAA, da Silva TC, Martins RP, Arantes LP, da Silveira TL, Dos Santos LV

[Neurochem Res, 2022]

Adenosine, a purine nucleoside with neuromodulatory actions, is part of the purinergic signaling system (PSS). Caenorhabditis elegans is a free-living nematode found in soil, used in biological research for its advantages as an alternative experimental model. Since there is a lack of evidence of adenosine's direct actions and the PSS's participation in this animal, such an investigation is necessary. In this research, we aimed to test the effects of acute and chronic adenosine at 1, 5, and 10mM on nematode's behaviors, morphology, survival after stress conditions, and on pathways related to the response to oxidative stress (DAF-16/FOXO and SKN-1) and genes products downstream these pathways (SOD-3, HSP-16.2, and GCS-1). Acute or chronic adenosine did not alter the worms' morphology analyzed by the worms' length, width, and area, nor interfered with reproductive behavior. On the other hand, acute and chronic adenosine modulated the defecation rate, pharyngeal pumping rate, and locomotion, in addition, to interacting with stress response pathways in C. elegans. Adenosine interfered in the speed and mobility of the worms analyzed. In addition, both acute and chronic adenosine presented modulatory effects on oxidative stress response signaling. Acute adenosine prevented the heat-induced-increase of DAF-16 activation and SOD-3 levels, while chronic adenosine per se induced DAF-16 activation and prevented heat-induced-increase of HSP-16.2 and SKN-1 levels. Together, these results indicate that exogenous adenosine has physiological and biochemical effects on C. elegans and describes possible purinergic signaling in worms.

Maria Catarina Silva et al. (2007) International Worm Meeting "Genetic modifiers of polyglutamine aggregation and toxicity in C. elegans."

Richard I. Morimoto, Susan Fox, Monica Beam, Margarida D. Amaral, Maria Catarina Silva

[International Worm Meeting, 2007]

Protein misfolding and aggregation are common characteristics of several human neurodegenerative diseases. Among these are the polyglutamine expansion disorders, a group of unrelated diseases that arise from the presence of polyglutamine stretches in the coding region of a specific gene. In order to study the cellular mechanisms underlying protein sequestration into aggregates and its consequences, we established a Caenorhabditis elegans model expressing different lengths of polyQ-YFP chimeras. To identify the cellular factors involved in polyQ aggregation, we performed an RNA interference screen for suppression of aggregation. We used a high throughput automated system and identified suppressors by looking for a reduction in the number of fluorescent foci. The genome-wide screen was performed using the threshold Q-length for aggregation, Q35, and it revealed 153 candidate modifiers. These candidates were then counter-screened for the Q37, and 67 common suppressors were identified. They are distributed among different functional classes: gene expression, protein synthesis and folding, transport, signaling, metabolic pathways and cell structure. To better understand the mechanisms involved in protein aggregation we will characterize how the decrease in aggregation is achieved: protein levels, protein biochemical and biophysical properties, pathways involved and alterations upon RNAi. We are also interested in determining how aggregation suppression affects toxicity. Population behavioral assays (using motility as a read-out for muscle dysfunction and toxicity) indicate that there isnt a direct correlation between suppression of aggregation and suppression in polyQ-induced toxicity. Some modifiers also increase organism toxicity while decreasing polyQ aggregation. Further studies will address how aggregation suppressors affect cellular integrity (structure) and homeostasis (using ts mutants as sensors for disruption of the folding environment). Study of the pathways involved in aggregation and identification of the toxic protein conformations, will elucidate the mechanism of misfolded proteins accumulation and allow its modulation to prevent toxicity.

Pires da Silva A et al. (2000) European Worm Meeting "Novel mutants defective in vulva induction in Pristionchus pacificus"

Sommer RJ, Pires da Silva A

[European Worm Meeting, 2000]

More than 50 genes involved in vulva development have been characterized in the rhabditid C. elegans. The extensive characterization of this model system provides a framework for studying the genetic basis of evolutionary modifications. Cell fate specification during vulva formation differs significantly between C. elegansand the diplogasterid nematode Pristionchus pacificus. Cell ablation experiments in P. pacificus demonstrate the existence of several novel cell-cell interactions (Sigrist and Sommer, 1999; Sommer and Sternberg, 1996). To understand the molecular mechanisms underlying vulval pattern formation in P. pacificus, we performed several TMP/UV and EMS mutagenesis screens. The mutants isolated in these screens fall into two categories: I) "generation-vulvaless" mutants do not generate Pn.p cells; II) "induction-vulvaless" mutants generate Pn.p cells, but these fail to differentiate. We have isolated and characterized a new class of induction vulvaless mutants, in which P6.p differentiates as a normal 1 cell, but P5.p and P7.p adopt a non-vulval fate (3). In wild type P. pacificus, as in C. elegans, P6.p adopts the 1 fate, whereas P5.p and P7.p generate the 2 lineage. Four mutants, tu114(TMP/UV), tu132(EMS), tu48(EMS) and tu51 (EMS), display this defect to different extents. Complementation test among these mutants are in progress. We are using a PCR-based genomic substraction strategy (RDA) to identify the deletion associated with the tu114 mutant. The identification of the genes involved in the induction of the vulva in P. pacificus may help us understand how molecular mechanisms change during evolution. Sigrist, C. B., and Sommer, R. J. (1999). Vulva formation in Pristionchus pacificus relies on continuous gonadal induction. Dev Genes Evol 209, 451-9. Sommer, R. J., and Sternberg, P. W. (1996). Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 6, 52-9.