Meredith Scheider et al. (2008) C.elegans Neuronal Development Meeting "A Genetic Screen to Identify Suppressors of the grk-2 Quinine Avoidance Defect"

Angela Chaparro-Garcia, Meredith Scheider, Denise Ferkey

[C.elegans Neuronal Development Meeting, 2008]

The C. elegans genome encodes over 1000 predicted G protein-coupled receptors (GPCRs), many of which are believed to be chemosensory. As a soil dwelling nematode that cannot see or hear, C. elegans must rely heavily on their ability to detect chemical cues to successfully navigate their environment. It is therefore essential that signaling through chemosensory GPCRs be carefully regulated. One mode of regulation is via G protein-coupled receptor kinases (GRKs), which specifically phosphorylate activated GPCRs to terminate signaling. Interestingly, despite the previously described role of GRKs in GPCR signal downregulation, C. elegans lacking grk-2 function are not hypersensitive to odorants. Instead loss of grk-2 broadly disrupts chemosensation and animals are unable to respond to a number of chemical stimuli including octanol (ASH), quinine (ASH), diacetyl (AWA) and isoamyl alcohol (AWC) (Fukuto and Ferkey et al., 2004). We have hypothesized that in the absence of grk-2 function there may be a compensatory down regulation of G protein signal transduction. RGS (regulator of G protein signaling) proteins are important negative regulators of GPCR signaling. RGS proteins act as GAPs (GTPase activating proteins) to accelerate the rate of GTP hydrolysis and dampen signaling through Galpha subunits. Loss of eat-16, which encodes an RGS protein, restores chemosensory response to diacetyl, detected by AWA, in grk-2 mutant animals (Fukuto and Ferkey et al., 2004). This suggested that RGS proteins may be important negative regulators of signal transduction. Because it appeared that EAT-16 may downregulate AWA chemosensory signaling in the absence of GRK-2, we sought to determine if loss of other RGS proteins could restore the chemosensory responses mediated by other sensory neurons in grk-2 mutant animals. To date, we have not identified another neuronally expressed RGS protein whose loss restores grk-2 mutant animals'' response to octanol or quinine (both detected primarily by ASH). To identify regulators of signaling that may be acting in the absence of grk-2 in the ASH sensory neurons, we performed a classical genetic suppressor screen. Using the grk-2 sensitized background, we screened 25,000 animals to identify mutations that restored the grk-2 response to the tastant quinine. We isolated 7 candidates that we have begun to characterize and map using standard mapping techniques. Our current mapping and behavioral characterization will be presented.

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.

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.

Materi WP et al. (1998) West Coast Worm Meeting "THE ROLE OF UNC-119 IN AXONOGENESIS IN C. ELEGANS"

Pilgrim DB, Maduro MF, Materi WP

[West Coast Worm Meeting, 1998]

UNC-119 is the first example of a novel class of protein found in both the developing and adult nervous systems of the nematode, the fly, the zebrafish (see poster by Angela Manning) and in mammals. While UNC-119 is found throughout the C. elegans nervous system, beginning around the 60-cell stage, the human and rat homologs (HRG4 and RRG4, respectively) have only been found in retinal tissue, beginning shortly after birth. The fly homolog (DmUnc-119) is expressed throughout the larval central nervous system. In the worm, four unc-119 null alleles have been characterized in addition to the wild type. Apart from the canonical nearly paralyzed, uncoordinated phenotype, these mutants also exhibit constitutive feeding behavior even in the absence of food and are unable to enter into the dauer larva stage when subjected to overcrowding and starvation. Both the behavioral and morphological phenotypes suggest that UNC-119 plays a role in growth cone guidance in the developing nervous system. An UNC-119::GFP fusion protein is found in the cytoplasm of all cells of the nervous system. In an unc-119 mutant the ventral nerve cord (VNC) is defasciculated and the lateral commisures, which normally project from the VNC dorsolaterally to join the dorsal nerve cord have an abnormal, highly branched structure. We have integrated GFP constructs, driven by promoters specific to subsets of neurons, into both wild-type and unc-119 mutant strains and compared neural structures at high magnification using confocal microscopy. The projections of many neurons are unaffected by the unc-119 mutation while abnormally long axons, abnormally branched axons and defasciculated axons are associated with neurons affected by the mutation. We have also conducted a yeast 2-hybrid screen using an unc-119 bait construct and have found several positive interactions that are selective for UNC-119 versus a SNF-4 bait. These preliminary results are presently being confirmed using the human homolog, HRG4, as bait in a 2-hybrid experiment. Co-immunoprecipitation experiments are also underway.

Gharib S et al. (2000) West Coast Worm Meeting "Cellular and genetic analysis of Gq mediated signaling pathways in C. elegans"

Gharib S, Simon MI, Sternberg PW, Bastiani CA

[West Coast Worm Meeting, 2000]

egl-30 encodes the C. elegans homologue of the mammalian heterotrimeric G protein alpha subunit, Gq. Reduction-of-function and loss-of-function mutations in egl-30 affect diverse behaviors in C. elegans. These include: viability, egg-laying, pharyngeal pumping, movement, and spicule protraction. In a screen for suppressors of the reduction-of-function mutation, egl-30(md186), two intragenic suppressor mutations were recovered. One of the mutations lies in a region in close proximity to residues involved in guanine ring binding. The other mutation is in a region that might affect receptor interaction, and is not expected to make contacts with the region that contains the original egl-30(md186) mutation. These mutants phenotypically resemble worms that overexpress egl-30, and have been used as a tool to characterize genes that function in a pathway with egl-30. From these studies, it is clear that EGL-30 regulates egg laying via downstream signaling pathways distinct from downstream pathways so far defined that control movement (1), viability (2), and spicule protraction (3). A rescuing gfp::egl-30 transgene confirms that egl-30 is coexpressed in some cells with egl-8 and lfe-1/itr-1/dec-4, two genes that encode signaling molecules defined as downstream of Gq in mammalian systems, and that appear to be downstream of egl-30 with respect to movement and viability, respectively (1,2). However, egl-30 is also uniquely localized and expressed in some cells. To determine the basis for the differences in genetic interactions with respect to different behaviors, we are develooping a system to characterize cell-type-specific molecular interactions with EGL-30 in vivo . 1. Lackner MR, Nurrish SJ, Kaplan JM; Neuron 24: 335-346 1999 2. Hajdu-Cronin YM and Sternberg PW, personal communication 3. Garcia LR and Sternberg PW, personal communication

Todd R Gruninger et al. (2004) West Coast Worm Meeting "Troponin T may act downstream of UNC-103 to regulate spicule muscle contraction in the C. elegans male."

L Rene Garcia, Todd R Gruninger

[West Coast Worm Meeting, 2004]

During male mating behavior, two types of protractor muscle contractions are evident, periodic contractions and tonic contraction. The dominant loss of function unc-103 (sy557) mutation causes about 60% of the males to undergo tonic muscle contraction in the absence of mating stimulation. UNC-103 is an ERG-like potassium channel that regulates contraction of the male sex muscles before and during mating. 1 It is expressed in many neurons in both the hermaphrodite and male, yet the only observable phenotype seen is constitutive contraction of the male spicule muscles; hermaphrodites appear normal. To identify pathways that are regulated by unc-103 , we EMS mutagenized unc-103 (sy557) hermaphrodites and screened for mutants that suppressed the sy557 -induced protraction. From this screen, we isolated lev-11 (rg1) , which reduced sy557 -induced protraction to 15%. The rg1 mutation in tropomyosin creates a missense mutation near the region of troponin T interaction. Troponin T (TNT) is a component of the tropomyosin/troponin complex that mediates calcium regulation of muscle contraction. 2 Protractor muscle contraction requires intracellular calcium from either extracellular pools or intracellular pools to activate the contractile apparatus. Voltage-gated potassium channels, such as UNC-103, are known to down regulate cell excitability, and thus down regulate voltage-gated calcium channels. Hence, the inappropriate muscle contraction seen with the unc-103 (sy557) background may be a result of abnormal extracellular calcium influx into the cytoplasm. We asked if the male-specific muscles have a calcium sensor that is inappropriately activated by the unc-103 (sy557) -induced mutation. To address this question, RNA Interference (RNAi) was used to reduce the four TNT genes in the male. Reducing TNT-4 at the L4 stage in males will suppress the unc-103 (sy557) -induced spicule contraction. A GFP fusion to TNT-4 was then constructed to visualize where in the male TNT-4 is expressed. GFP fluorescence was seen only in the pharynx. We are currently investigating the site of action of TNT-4 with tissue specific rescues of tnt-4 (gk136) to verify that a reduction in a troponin component only required in the pharynx indeed suppresses sy557 -induced spicule contraction. 1. Garcia and Sternberg. 2003 Apr 1. J Neurosci . 23(7):2696-705. 2. Potter et al. 1995. J. Biol. Chem. 270:2557-2562.