Boucher, Benjamin et al. (2015) International Worm Meeting "From genetic interaction modules to robustness and evolution of a multicellular organism."

Boucher, Benjamin, Hallett, Michael, Lee, Anna, Jenna, Sarah

[International Worm Meeting, 2015]

A genetic interaction (GI) between two genes is defined when the genetic alteration of one gene modifies the phenotypic expression associated with the alteration of a second gene. Genome-wide efforts to map GIs in yeast revealed structural properties of a GI network, providing insights into the mechanisms underlying the robustness of yeast to genetic and environmental insults, and also into the link existing between genotype and phenotype. While GIs appear to be highly conserved between distant yeast species, conservation of GIs and GI network structures between unicellular and multicellular organisms is less evident. Characterization of the structure of the GI network in these later organisms is consequently of high interest. In this study, we present an in-depth characterization of ~1.5K GIs in the nematode Caenorhabditis elegans. We identified and characterized six distinct classes of GIs by examining a wide range of properties of the underlying genes and network structure including co-expression, phenotypical profiles, relationship with protein-protein interaction dense subnetworks (PDS) and pathways, molecular and biological functionalities, gene essentiality and pleiotropy. Our study highlights significant differences between GI relationships with PDS and pathways. We also established that specific classes of GIs define two types of functional modules: PDS-centric and PDS-independent modules with low and high pleiotropic indices, respectively, as well as pleiotropic and non-pleiotropic connectors. Our study provides the first in-depth characterization of a genetic interaction network in a multicellular organism. It also suggests how the modular structure of this network contributes to system robustness and evolution.

Cipriani, Patricia G. et al. (2013) International Worm Meeting "A genome-wide network of genetic interactions in embryonic development."

Erickson, Katherine, Cipriani, Patricia G., White, Amelia, Piano, Fabio, Gunsalus, Kristin, Kao, Huey-Ling, Reboul, Jerome, Munarriz, Eliana, Lucas, Jessica, Chatterjee, Indrani

[International Worm Meeting, 2013]

The phenotypes manifested by genetic alleles are influenced by the genetic background in which they reside. Yet, we still have a very limited understanding of how genetic interactions (GIs) influence animal development. The goal of our project is to use genome-wide screens to identify all enhancing and suppressing GIs for a set of strains harboring temperature sensitive (ts) mutations in 24 essential embryonic genes. We have completed over three million primary GI assays and secondary screening of putative suppressors, and we have archived in a database all experimental metadata and images, along with quantitative scoring results from an automated phenotypic scoring algorithm we developed (DevStaR). DevStaR combines computer vision and machine learning methods to count different developmental stages in mixed populations of animals. Using these results we have developed a quantitative phenotypic "GI score" based on the multiplicative model of independence: if the effects of perturbing two genes are independent, then their combined effects should not deviate from the product of their individual effects. GI scores for individual experimental replicates correlate positively with semi-quantitative manual estimates of interaction strength. Using manual inspection as a reference, we devised criteria to combine GI scores across replicates that reliably detect suppressing interactions. We then generated final interaction scores that reflect both strength and reproducibility, which we used to define ~800 high-confidence and ~750 intermediate-confidence suppressing interactions. Based on comparisons with manual scoring, we estimate the false discovery rates in these two sets as 2% and 10%, respectively. The resulting GI network provides the first genome-wide map of suppressing genetic interactions for the embryo based on quantitative phenotypic analysis of viability.

Dayalan G Srinivasan et al. (2003) International Worm Meeting "A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function"

Ridgely M Fisk, Sander Van Den Heuvel, Huihong Xu, Audrey Perreault, Dayalan G Srinivasan

[International Worm Meeting, 2003]

The C. elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the meiotic and mitotic spindles. To identify components that act in these processes with LIN-5, we purified LIN-5 associated proteins from C. elegans embryos using large-scale immunoprecipitations and tandem mass spectrometry.This approach identified two nearly identical genes, gpr-1 and gpr-2 (G Protein Regulator), that act in spindle and chromosome movements, similar to lin-5. GPR-1/GPR-2 associated with LIN-5 in vivo and directly interacted with LIN-5 in vitro. Furthermore, GPR-1/GPR-2 colocalized with LIN-5 to the mitotic spindle and cell cortex in a lin-5-dependent manner.The predicted GPR-1 and GPR-2 proteins each contain a GoLoco/GPR motif that has been shown to interact with the Gi/o subunit of heterotrimeric G proteins. GOA-1 Gi/o colocalized with GPR-1/GPR-2 and LIN-5 at the cell cortex, and GPR-1 bound GOA-1 in a GDP-dependent manner. Inactivation of lin-5, gpr-1/gpr-2 or the Gi/o genes goa-1 and gpa-16 all caused highly similar cell division and spindle positioning defects, indicating a positive role for GPR-1/GPR-2 and LIN-5 in G protein signaling.GoLoco proteins have been suggested to primarily activate G signaling. However, gpr-1/gpr-2 inactivation did not phenocopy loss of G activity, but mimicked the loss of Gi/o activity. These and other experiments suggest that the LIN-5/GPR complex positively regulates G signaling.Together, these results indicate that LIN-5 localizes or recruits GPR-1/GPR-2 to the spindle and cortex, and in turn, initiate G signaling to affect spindle forces. Polarity determinants may regulate LIN-5/GPR/G locally to create the asymmetric forces that drive spindle movement. Additionally, ric-8 Synembryn was shown previously to regulate embryonic spindle position and genetically interact with goa-1. The mammalian homolog has been demonstrated to catalyze exchange of GDP for GTP in G, consistent with a two step model for receptor-independent activation of G signaling.

Youngmi Shin et al. (2010) East Asia Worm Meeting "Multiple pathways of ERK1/2 activation by the C. elegans muscarinic acetylcholine receptor GAR-3 in mammalian cultured cells"

Yang-Seo Park, Seungwoo Kim, Nam Jeong Cho, Youngju Shin, Youngmi Shin

[East Asia Worm Meeting, 2010]

Extracellular signal-regulated kinase 1/2 (ERK1/2) mediate diverse cellular functions, including cell proliferation, differentiation, and migration. ERK1/2 are evolutionary conserved enzymes in eukaryotes, and ERK1/2 activation is induced by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). It has been reported that the C. elegans G protein-linked acetylcholine receptor GAR-3 regulates feeding, mating, and locomotion. We have previously identified that GAR-3 activates ERK1/2 via phospholipase C/protein kinase C/MEK in Chinese hamster ovary (CHO) cells. In this study, we observed that GAR-3-mediated ERK1/2 activation was inhibited by the Gi/o protein inhibitor pertussis toxin (PTX), Src family kinase inhibitor PP1 analog, and phosphoinositide 3 (PI3) kinase inhibitor LY294002. These observations suggest that GAR-3 activates ERK1/2 through Gi/o protein, Src family kinase, and PI3 kinase. In human embryonic kidney (HEK) 293 cells, the ERK1/2 activation was reduced by epidermal growth factor receptor (EGFR) inhibitor PD168393 and a dominant negative Ras mutant, implying that EGFR and Ras are also involved in GAR-3-mediated ERK1/2 activation. Taken together, these results indicate that GAR-3 mediates ERK1/2 activation through multiple pathways.

Elizabeth B Rex et al. (2005) International Worm Meeting "Caenorhabditis elegans contains multiple tyramine receptors"

Elizabeth B Rex, Hong Xiao, Katy Smith, Richard W Komuniecki, Vera M Hapiak, Robert J Hobson

[International Worm Meeting, 2005]

Tyramine appears to regulate a number of key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, we identified a Caenorhabditis elegans tyramine receptor, SER-2, that was involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that also encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind [3H]-tyramine with high affinity with a Kd of 20 5 nM. Other physiologically relevant biogenic amines, such as octopamine and dopamine, inhibit [3H]-tyramine binding with much lower affinity (Kis of 1.55 0.5 and 1.78 0.6 mM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPS binding to membranes from cells expressing TYRA-2 (EC50 of 50 13 nM) and the TA-dependent GTPS binding is PTX-sensitive suggesting that TYRA-2 may couple to Gi/o. Based on fluorescence from tyr-2::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal, in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Gi/o-coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.

Ishihara T et al. (1997) International C. elegans Meeting "METABOTROPIC GLUTAMATE RECEPTORS IN C. ELEGANS"

Francesconi A, Katsura I, Ishihara T, Duvoisin RM

[International C. elegans Meeting, 1997]

Glutamate receptors are classified into ionotropic receptors and metabotropic receptors (mGluRs). mGluRs, which couple with G-proteins, are divided into three subgroups. The receptors in the first subgroup are coupled to Gq and the IP3/Ca2+ signal transduction system, while the other receptors are involved in inhibiting cAMP formation through Gi. By studying the knockout mice lacking the mGluR gene, it was shown that the mGluRs are involved in higher order functions in mammalian nervous systems, as well as in neural network formation. In the C. elegans genome project, two mGluR genes (mgl-1, mgl-2) have been identified. The predicted amino acid sequences show MGL-1 belongs to the Gi coupled mGluR group, while MGL-2 belongs to the Gq coupled group. Indeed, we found that the HEK293 cells expressing the mgl-2 cDNA increase in phosphoinostitol turnover, responding to glutamate. To elucidate the expression pattern of the C. elegans mGluR genes, we generated transgenic worms carrying a chimeric mGluR-GFP fusion gene. The fusion gene of mgl-2 is expressed essentially in interneurons, while that of mgl-1 is expressed in motorneurons and pharyngeal neurons as well as interneurons. To elucidate functions of mGluRs in C. elegans, we isolated mutants of mGluR genes by the Tc1 insertion/ deletion strategy. We examined various behavioral analyses of them. It seems that the mgl-2 mutant shows abnormal head movement and stronger tap reversal reflexes. We are testing whether these abnormalities can be rescued by the wild type mgl-2 gene.

G Jansen et al. (1999) International C. elegans Meeting "The complete family of G protein genes of Caenorhabditis elegans"

RH Plasterk, M van der Horst, E Hazendonk, KL Thijssen, G Jansen, P Werner

[International C. elegans Meeting, 1999]

One of the new possibilities in post-sequence genetics is the analysis of complete gene families at once. We studied the family of heterotrimeric G proteins. C. elegans has 20 G a , 2 G b and 2 G g genes. There is one clear homologue of each of the four mammalian classes of G a genes, Go/Gi, Gs, Gq, G12, and there are sixteen new a genes. While the conserved G a subunits are expressed in many neurons and muscle cells, GFP fusions indicate that 14 of the new G a genes are expressed almost exclusively in a small subset of the amphid cells, sensory neurons in the head of the nematode, or other putative sensory neurons. We isolated null alleles for all G a genes, using target selected gene inactivation. Furthermore gain-of-function alleles were generated by introducing the wild type genes as transgenes (XS). None of the amphid expressed genes are essential for viability. Based on the specific expression of many G a subunits in chemosensory neurons, all mutants were tested for chemotaxis to water soluble and volatile attractants and repellents. Only four loss-of-function alleles show chemotaxis defects. In contrast, nine gain-of-function alleles show altered behavior in the various chemotaxis assays. The lack of loss-of-function phenotypes can be explained by functional redundancy, as has been described for gpa-2 and odr-3 (Roayaie et al., Neuron 20: 55-67, 1998). Alternatively, the G a genes may be involved in the perception of compounds that have not yet been tested. Furthermore, they may have negative regulatory functions, as was found for gpa-5 and gpa-6 . We are further testing these hypotheses by combining several of loss-of-function alleles. Based on functional analysis the 20 G a genes can be divided into two groups: subunits that affect muscle activity (homologues of Gi/Go a , Gs a and Gq a ), and 14 new G a genes that are probably all involved in perception (Jansen et al., Nature Gen. In press).

Brundage LA et al. (1997) International C. elegans Meeting "gpa-12, A Gene Encoding a G12 Alpha Subunit"

Korswagen HC, Brundage LA, Simon MI, Plasterk RHA, Sternberg PW

[International C. elegans Meeting, 1997]

Heterotrimeric G proteins are classified into four families, Gi/Go, Gq, Gs and G12, based on similarity of their sequence and function. In contrast to the other G protein families, the receptors and the second messengers involved in transduction of signals via the G12 family are largely unknown. To elucidate the G12 pathway in C. elegans, we are studying the gene gpa-12, encoding a G12 alpha subunit on cosmid F18G5. We have engineered a constitutively active allele of gpa-12 under hsp-16 control. When this allele is induced by heat shock during embryogenesis, animals hatch into uncoordinated, developmentally arrested larvae which eventually recover. We plan on exploiting this phenotype to identify gpa-12 effectors in a genetic screen. We have also obtained a presumptive null allele of gpa-12, via the transposon insertion/ deletion method, which removes 90% of the gpa-12 coding sequence. Animals homozygous for this allele exhibit no obvious developmental or behavioral defects, a surprise since gpa-12::gfp is expressed broadly in the C. elegans musculature, as well as in a subset of the nervous system.

van der Hoeven R et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The Role of Dual Oxidase Ce-Duox1/BLI-3 in C. elegans Innate Immunity"

Chavez V, Garsin D A, Fellers M, van der Hoeven R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Initially ROS (reactive oxygen species) were thought to originate through an unspecific process as a byproduct from the mitochondrial respiratory chain. In recent years studies have demonstrated that ROS are produced in a specific and controlled manner by the NOX/DUOX family of proteins. These proteins are the sources of two species of ROS, mainly superoxide and hydrogen peroxide that participate in microbial killing, cell signaling and hormone biosynthesis. In mammals, DUOXs have been identified in the oral cavity, airways and the mucosal surfaces of the lower GI tract implicating a role in innate immune function. Furthermore it was shown that the ROS produced by DUOX has an antimicrobial function in the gut of Drosophila. C. elegans represents one of the simpler host organisms in which mechanisms of host defense can be dissected. Two DUOX homologues Ce-Duox1/BLI-3 and Ce-Duox2 have been identified in C. elegans. The process of tyrosine cross-linking in the cuticle is the main function of Ce-Duox1/BLI-3. Here we identify an additional role for Ce-Duox1/BLI-3 in ROS generation during immune defense against human pathogens such as E. faecalis and P. aerugionsa. Data visualizing the reaction in live animals observes ROS production occurring in the infected intestines. Reduction of Ce-Duox1/BLI-3 in the intestine and the hypodermis increases susceptibility to the pathogens. Current studies are focused on identifying how ROS production via Ce-Duox1/BLI-3 is regulated in response to infection.

E Cuppen et al. (1999) International C. elegans Meeting "Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies"

G Jansen, RH Plasterk, E Cuppen

[International C. elegans Meeting, 1999]

Heterotrimeric G proteins are signal transduction molecules situated at the interface between extracellular signals received via G protein-coupled receptors (over 600 predicted in the C.elegans genome) and intracellular second messenger systems. We set out to study the function of all heterotrimeric G protein genes in C.elegans . Thus far, two G b subunit genes ( gpb-1 and 2 ), two G g genes ( gpc-1 and 2 ) and 20 G a genes have been identified in the genomic sequence (Jansen et al., Nature Gen. in press ). Besides one member ( gsa-1 , goa-1 , egl-30 and gpa-12 ) of each of the mammalian G a classes (Gs, Go/Gi, Gq, and G12) there are 16 new C.elegans specific genes ( gpa-1 to 11 , 13 to 16 and odr-3 ). We have cloned the cDNA's for all the G protein subunits. RT-PCR and sequence analysis gave no indications for alternative splicing, but we found that about 10% of the exons of this highly conserved protein family were predicted incorrectly by Genefinder. We will employ all cDNA's in yeast two- and three-hybrid interaction studies to test for interaction specificity between the different subunits. Furthermore, we will screen cDNA libraries for interacting proteins. The interaction specificity of novel proteins can easily be tested using mating assays, because all C.elegans heterotrimeric G protein subunits are available. Since we know that multiple G a subunits are expressed in particular cells (Jansen et al., Nature Gen. in press ), specificity in interactions mediated by these subunits may provide an efficient mechanism regulating signaling via G protein coupled receptors.