Jose, Antony M. et al. (2009) International Worm Meeting "C. elegans tissues do not require RNA interference to generate and export silencing signals derived from expressed RNAs."

Hunter, Craig P., Garcia, Carlo, Jose, Antony M., Smith, Jessica J.

[International Worm Meeting, 2009]

Double-stranded RNA (dsRNA) triggers RNA interference (RNAi) to silence genes of matching sequence. RNAi-like mechanisms underlie key cellular processes such as heterochromatin formation, regulation of gene expression, and silencing of repetitive DNA. In some animals, when exogenous dsRNA is introduced into a cell, the resultant silencing is transported to other cells. However, numerous dsRNAs and hairpin RNAs (hpRNAs) are transcribed from animal genomes but whether expressed RNAs and endogenous RNAi-like mechanisms generate mobile silencing signals is unknown. Studies in C. elegans have shown that the conserved dsRNA channel Systemic RNAi Defective-1 (SID-1) is required for the import of transported silencing signals. We found that the efficient silencing of multicopy transgenes requires SID-1, suggesting that transgene silencing in one cell produces mobile silencing signals that function to initiate and/or maintain transgene silencing in another cell. Further, the tissue-specific expression of RNAi triggers resulted in the transport of silencing from all tested tissues to other tissues, consistent with expressed RNAi triggers generating mobile silencing signals. Although import through SID-1 suggests that mobile silencing signals are likely forms of dsRNA, their precise identity and biogenesis are unknown. We reasoned that proteins that act on dsRNA for RNAi within a cell may modify dsRNA or other dsRNA-derived RNAs to generate mobile silencing signals. Therefore, we examined the role of RNAi Defective-1 (RDE-1), an Argonaute protein required for RNAi in C. elegans, in the export of mobile silencing signals. In rde-1(-) animals, when gfp-dsRNA was expressed in the pharynx, no silencing of GFP expression was detected in either the pharynx or the body wall muscles. However, when rde-1(+) was expressed specifically in the body wall muscles of these animals, gfp expression was silenced only in that tissue. Consistent with the export of mobile silencing signals from an rde-1(-) pharynx, GFP expression in the pharynx remained unaffected. Thus, RNAi-mediated degradation of target mRNA is not required for the generation and export of mobile silencing signals derived from transgenically expressed RNAi triggers. We are systematically testing additional components and regulators of the RNAi pathway for possible roles in the generation of mobile silencing signals.

Barbieri, Sofia et al. (2021) International Worm Meeting "The dynamic partnered dance between PLK-1 and MEX-5: interpreting gradient formation with computational modelling."

Gotta, Monica, Barbieri, Sofia, Griffin, Erik E., Nurni Ravi, Aparna

[International Worm Meeting, 2021]

In C. elegans embryos, PLK-1 is pivotal for cell division and it orchestrates polarity establishment together with its binding partner, MEX-5. To achieve this, their localization/activity must be precisely regulated. MEX-5 enrichment at the anterior cytoplasm results from a change in its diffusivity following uneven phosphorylation along the embryo axis. We know PLK-1 relocalization to the anterior depends on MEX-5. However, the biological and physical mechanisms behind the dynamics of this protein are still poorly described. To address this, PLK-1 and MEX-5 gradient formation was measured in two CRISPR strains and significant discrepancies were revealed between the two proteins in terms of: 1) gradient steepness, as PLK-1 forms a less steep gradient compared to MEX-5; 2) dynamics, with PLK-1 gradient establishment delayed and slower; 3) diffusivity, as PLK-1 diffusion coefficient does not correspond to MEX-5's one from anterior to posterior. To shed light on PLK-1 dynamics, and how it is intertwined to MEX-5, we developed a novel Monte Carlo simulation framework able to recreate the protein motions in the C. elegans one-cell embryo. Thanks to our computational approach, we were able to postulate on the biological mechanisms behind MEX-5 and PLK-1 dynamics during the whole cell division, from early embryos to the steady-state before cytokinesis. The simulations succeed in reproducing PLK-1 gradient formation, in agreement with experimental measurements, if: 1) PLK-1 binds to phosphorylated MEX-5; 2) the binding is triggered after a defined time delay; 3) PLK-1 dynamically interacts with MEX-5, leading to a continuous replenishment of a pool of unbound PLK-1. The Monte Carlo framework we propose can eventually be applied to other polarity-related factors or mutants in which polarization is perturbed, to understand if it can be traced back to a failure in PLK-1 localization. Finally, conditions where gradient formation is altered, like after stress, can be simulated.

Dengg M. et al. (2006) European Worm Meeting "Using Caenorhabditis elegans to Study Cellular Responses to Endogenous DNA Damage"

Boulton S., Nilsen H., Garcia-Muse T., Dengg M.

[European Worm Meeting, 2006]

Dengg, M. 1, Garcia-Muse, T.2, Boulton, S.2, and Nilsen, H1 DNA repair pathways and intracellular signalling cascades contribute to DNA damage response (DDR) mechanisms that determine cellular responses to DNA damage. C.elegans has been used successfully to study DDR in response to DNA damage induced by physical or chemical agents. Whether DDR is activated in response to the most abundant forms of DNA damage, arising spontaneously through the reaction of water and reactive oxygen species with DNA, is poorly understood. Compared with treatment with physical or chemical DNA damaging agents, the study of DDR in response to endogenous lesions must overcome technical problems with respect to inducing sufficiently high levels of DNA lesions to allow studies of downstream responses. We have exploited RNA interference (RNAi) technology in C.elegans to modulate dUTP pools in order to achieve incorporation of uracil above background levels. Here, we present data describing the phenotypical consequences of increased incorporation of uracil into DNA. The role of DNA repair and DNA damage checkpoints in activating DNA damage response pathways in response to uracil will be presented.

Elise Walck et al. (2008) Development & Evolution Meeting "A Glazier-Granier-Hogeweg (GGH) Model For Caenorhabditis elegans Embryonic Development"

Scott Thatcher, Clayton Davis, Timothy Walston, Elise Walck

[Development & Evolution Meeting, 2008]

An understanding of the forces acting on cells in the early embryo can provide important information for how cells interact to determine their shapes, movements and fates. These forces can be both physical and genetic and can be intracellular or extracellular. The early Caenorhabditis elegans embryo provides an excellent environment to explore the forces acting during embryogenesis and to develop techniques that can be applied later to more advanced biological events. We report the development of a 4D GGH (Glazier-Granier-Hogeweg) model to simulate the 4-cell stage of embryogenesis. GGH modeling uses a Metropolis Monte Carlo algorithm to describe the evolution of systems based on the idea that many systems transform to minimize their overall energy. By using a fixed-lattice environment governed by the Hamiltonian, an overall energy equation, GGH modeling can simplify the description of cell-based phenomena. The early stages of embryogenesis offer an environment where components of the Hamiltonian can be identified and tested. Our Hamiltonian includes cell-cell adhesion, cell-shell contact, centromere rotation and elongation, and constraints of surface area and volume. Our GGH model allows for the incorporation of several biological components that have not be explored in previous models of C. elegans embryogenesis. In addition, several aspects of our model broaden the possible applications of GGH modeling.

Hills, A.J. et al. (2017) International Worm Meeting "Markov chain Monte Carlo simulation of the dauer larvae formation decision-making and signalling in populations of Caenorhabditis elegans."

Hills, A.J., Harvey, S.C., Viney, M.E.

[International Worm Meeting, 2017]

Phenotypic plasticity allows species to respond to environmental changes. In the wild, populations of the nematode Caenorhabditis elegans develop and grow in nutrient-rich, ephemeral habitats. Movement between these ephemeral patches, and long term genotype survival, depends on the development of dauer larvae. Within these natural populations, survival is therefore dependent upon a critical development decision, as worms must commit to either a reproductive fate to increase local numbers, or a migratory fate to find new resources to exploit. Failure to disperse at the correct time will result in loss of the local population with the resources in the ephemeral habitat. Under laboratory conditions, the decision between dauer and non-dauer larval development is driven by ascaroside signalling - which acts as a proxy for population size - by food availability, and by temperature. However, phenotypic differences between genotypes in reaction to these factors is substantial. For example, ascaroside production profiles vary between genotypes as do the responses to specific ascarosides and to mixtures of ascarosides. There is also evidence that suggests that ascaroside signalling by worms may be manipulative. Here we present the results of Markov Chain Monte Carlo (MCMC) simulations of modelled C. elegans genotypes optimising the developmental switch under different environmental pressures. The simulation results are compared with a number of wild-type strains. The evolution of ascaroside signalling is examined with the additional effects of exposure to signal distortion and noise as may be present in heterogeneous wild habitats. The model is presented in the form of a generalizable method for studying developmental decisions and other phenotypically plastic traits under a variety of environmental conditions.

Teixeira, J. et al. (2019) International Worm Meeting "Identification of the ubiquinone pathway as a suppressor of Myotonic Dystrophy-associated toxicity."

Brandao, A., Garcia, S., Teixeira, J., Harju, A.

[International Worm Meeting, 2019]

Myotonic dystrophy type 1 (DM1) is the most common type of dystrophy in adulthood. It is caused by the accumulation of mutant RNA in the nucleus due to the expression of expanded CUG repeats in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene. These RNA-bearing CUG repeats form hairpin structures that interact inappropriately to RNA binding proteins such as splicing factors, namely muscleblind-like (Mbnl) and CUG-binding protein (CUGBP) families, thereby causing aberrant alternative splicing leading to a multisystemic disease. Notably, due to the multisystemic nature of DM1, the full extent of cellular processes affected by these toxic RNAs is still unknown. Our aim is to identify new modulators and mechanisms of RNA toxicity. To address this aim, we performed an RNAi screen by using a previously characterized C. elegans DM1 model[1]. Since this C. elegans model mimics DM1 phenotypes, changes in its motility defect were used as readout for toxicity. We identified the ubiquinone (CoQ) pathway as a suppressor of DM1, as downregulation of this pathway increases DM1 toxicity, whereas CoQ supplementation partially rescues the DM1 motility defect. Furthermore, our data also suggest that complex II of mitochondrial electron transport chain is implicated in DM1 dysfunction. The role of mitochondria in DM1 pathogenesis is further underlined by our preliminary results showing that DM1 animals have an altered mitochondrial morphology. Taken together, we established a genetic connection between DM1, ubiquinone pathway and mitochondrial function and are currently examining the mechanisms of DM1 mitochondrial dysfunction regulation. 1. Garcia, S.M., et al., Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans. Nat Struct Mol Biol, 2014. 21(8): p. 712-20.

Ellen Nollen et al. (2006) European Worm Meeting "C. elegans Models of Protein Misfolding Diseases"

Ellen Nollen, Tjakko van Ham, Ronald H. A. Plasterk.

[European Worm Meeting, 2006]

Ellen Nollen, Tjakko van Ham & Ronald H. A. Plasterk. Aggregation of misfolded proteins occurs in various age-related neurodegenerative disorders, including Parkinsons, Alzheimers, and Huntingtons disease. To understand how cells protect themselves against misfolded proteins, we search for genes that enhance or prevent protein aggregation. C. elegans strains expressing polyglutamine stretches fused to YFP with visible, age-dependend protein aggregation are used as a genetic model. Using a genome-wide RNAi screen, we have previously identified 186 genes that, when knocked down, cause premature protein aggregation. These genes include genes involved in protein synthesis, folding, degradation and RNA synthesis and processing. 1. Conversely, we performed a forward mutagenesis screen to identify genes that, when mutated, suppress age-dependent polyglutamine aggregation. For one suppressor mutant, in which aggregation is suppressed by more than 75%, we have now identified the responsible mutation. This mutation is a missense mutation in a gene encoding a protein of unknown function that is highly conserved between C. elegans and humans. Knock-down by RNAi of the same gene in wild-type worms yielded a similar reduction in aggregation, suggesting a loss-of-function mutation. We are currently further characterizing this mutant and the remaining suppressor mutants. In addition, to establish whether the genes we have identified are specific for polyglutamine aggregation or whether they comprise of a general protein homeostatic buffer, we have developed a worm model for aggregation of alpha synuclein, which occurs in Parkinson''s disease. Altogether our results will provide insight into cellular protection against misfolded proteins and yield targets for therapy against protein misfolding diseases.. 1Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.

Guo, Xiaoyan et al. (2013) International Worm Meeting "SIR-2.1, an HDAC, is required to maintain male mating ability during aging of C. elegans."

Garcia, Luis Rene, Guo, Xiaoyan

[International Worm Meeting, 2013]

C. elegans males display a significant deterioration of mating behavior during 'early aging' prior to the structural dysfunction of neuromuscular circuitry. The mating deterioration is correlated with an increase of the excitability in the male mating circuitry(1). Here, we showed that the mating behavior of males with sir-2.1 null mutation declines even prematurely compared to that of wild-type males, and the mating circuitry of sir-2.1(ok434) is more excitable than that of wild type males. Through Ca2+ imaging in mating males, we demonstrated that the hyper excitation of sex muscles during mating blocks the process of sperm transferring in 2-day-old sir-2.1(ok434) males, hence leading to the failure of mating. Furthermore, we illustrated that sir-2.1(ok434) males generate more reactive oxygen species (ROS) possibly due to enhanced catabolism in the mating circuits and/or reduced ability to scavenge ROS because of reduced expression of ROS-scavenger genes such as superoxide dismutase sod-1and glutathione transferase gsto-1. Meanwhile, we found that artificially increasing ROS stress by feeding males with paraquat elevates the excitability of the mating circuitry and reduces the mating potency. In addition, reducing ROS by feeding males with N-Acetyl Cysteine (NAC), an antioxidant reagent, lowers the excitability of the mating circuitry and improves mating. Taken together, we conclude that SIR-2.1, a histone deacetylase (HDAC) regulates the physiological state of the mating circuitry possibly through regulation of the oxidative stress. Reference: 1.Guo X, Navetta A, Gualberto DG, Garcia LR. Behavioral decay in aging male C. elegans correlates with increased cell excitability. Neurobiol Aging. 2012.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Rodrigues, Pedro Reis et al. (2009) International Worm Meeting "A variety of age-dependant aggregating proteins determine lifespan."

Peters, Theodore, Gibson, Brad, Lithgow, Gordon, Hughes, Robert, Alavez, Silvestre, Rodrigues, Pedro Reis, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Protein aggregation has for long been hypothesised as a determinant of lifespan. Briefly, normal cellular activity may give rise to damaged proteins causing them to become insoluble, missfold and aggregate. To test this hypothesis we adapted a protocol in order to extract insoluble proteins from synchronously aging populations of C. elegans. Proteins were separated based on their aqueous and detergent solubility and the insoluble fraction was resolubilized in 70%; formic acid. Insoluble proteins were chemically labelled, identified and quantified by liquid chromatography coupled with mass spectrometry (LC- ESI-MS/MS). We identified a range of proteins with roles in various cellular processes and possibly from a range of cellular compartments. 27%; of the proteins identified as forming aggregates have previously been shown to be important in keeping low levels of polyglutamine aggregation1. This suggests that reduced soluble levels of these proteins caused by age-related aggregation may cause increased risk of polyglutamine aggregation. We then considered whether proteins that appear to form aggregates during normal aging influenced lifespan. To test this notion we, reduced their expression in adult animals (from 4 days old) by RNA interference (RNAi). 34%; of the RNAi treatments were found to significantly extend mean lifespan in C. elegans suggesting that a variety of age-dependant aggregating proteins determine lifespan. Among the insoluble proteins, DAF-21, an ortholog of the mammalian HSP-90, showed age-dependant aggregation and is being used as a marker to study the role of several molecular pathways in protein aggregation. Taken together our results suggest that protein aggregation may play a common and key role in aging and age-related disease. 1 - Nollen, E., Garcia, S., Haaften, G., Kim, S., Chavez, A., Morimoto, R., Plasterk, R., Genome-wide RNA interference screen identified previously undescribed regulators of polyglutamine aggregation. Proc Natl. Acad. Sci., 101, 6403-6408, 2004.