Z Gitai et al. (1999) International C. elegans Meeting "The netrin UNC-6 can redirect axons in vivo"

CI Bargmann, M Tessier-Lavigne, Z Gitai

[International C. elegans Meeting, 1999]

The netrin UNC-6 is involved in both the attraction and repulsion of circumferentially migrating cells and axons in C. elegans. Attraction to netrin/UNC-6 is mediated by the UNC-40 receptor, while repulsion is mediated mainly by the UNC-5 receptor. Culotti et al. have performed extensive characterization of the genetic pathways for UNC-5-dependent netrin repulsion, but less is known about UNC-40-dependent netrin attraction. In wild type animals UNC-6 is expressed ventrally, and the HSN axon is directed ventrally via the UNC-40 receptor, which can act at least partially cell autonomously within the HSN neuron to mediate ventral guidance. This ventral migration is severely disrupted in unc-6 and unc-40 mutants. We used the muscle-specific fragment of the UNC-129 promoter (kindly provided by Drs. A. Colavita and J. Culotti) to misexpress UNC-6 in the dorsal muscle. In an unc-6 mutant background, HSN axons can be redirected toward the dorsal UNC-6 source. This demonstrates that axons can be attracted to UNC-6 in vivo . The attractive migration toward both endogenous and ectopic UNC-6 requires the UNC-40 receptor. To identify additional genes required for UNC-6 attraction, we are currently screening for mutations that suppress the HSN redirection to dorsal UNC-6. These genes are expected to define components of the UNC-40 pathway. Axonal growth cones are exposed to many different cues that act in concert to direct their trajectories. Guidance receptors such as SAX-3/Robo normally act in parallel to UNC-40 to direct ventral HSN axon guidance. In animals with ectopic dorsal expression of UNC-6, the dorsal migration of the HSN axon is directed solely by UNC-6. Other pathways that direct HSN ventral guidance are not expected to be affected by the dorsal UNC-6, and continue to direct the HSN axon ventrally. Thus, these pathways would serve to antagonize HSN attraction to the ectopic dorsal UNC-6. Mutations in components of these pathways should present themselves as enhancers of the redirection to dorsal UNC-6. The dorsal UNC-6 strain therefore provides a useful genetic background to identify and study molecules required for the reception and transduction of the UNC-6/UNC-40 signal, as well as parallel pathways that specify ventral axon guidance.

Gitai Z et al. (1998) West Coast Worm Meeting "UNC-6 CAN REDIRECT AXONS IN VIVO"

Bargmann CI, Tessier-Lavigne M, Gitai Z

[West Coast Worm Meeting, 1998]

The UNC-6 netrin is thought to be involved in both the attraction and repulsion of circumferentially migrating cells and axons. Consistent with that idea, such migrations are disrupted in unc-6 mutants. It has also been shown that in vitro, the UNC-6 vertebrate homolog, Netrin-1, is sufficient to elicit both axonal outgrowth and turning. However, it has yet to be clearly demonstrated whether UNC-6 can play an instructive role in directing axon migrations in vivo. To address that question we have focused on the HSN motor neuron. In wild type animals UNC-6 is expressed ventrally, and the HSNUs initial axon projection is directed ventrally. The disruption of this ventral migration is one of the most penetrant defects associated with unc-6 mutants. We used the muscle-specific fragment of the UNC-129 promoter (kindly provided by Joe Culotti) to misexpress UNC-6 in the dorsal muscle. In an unc-6 mutant background, where the only source of UNC-6 is the ectopic dorsal source, the HSN axons can be misdirected dorsally. This shows that axons can be redirected to grow toward an ectopic source of UNC-6 in vivo. The attractive migration towards UNC-6 requires the UNC-40 receptor. Little is known, however, about the mechanism by which UNC-6Us interaction with UNC-40 signals to the cyctoskeleton to elicit directed migration. Axon growth cones are normally exposed to many different signals directing their growth. In worms with ectopic dorsal UNC-6, we appear to have migrations directed solely by UNC-6. This strain can now be used to identify and study molecules required for presentation, reception, and transduction of the UNC-6 / UNC-40 signal.

Gitai Z et al. (2000) West Coast Worm Meeting "Cytoskeletal Signalling in Response to the UNC-6 Axonal Attractant"

Bargmann CI, Lundquist EA, Tessier-Lavigne M, Gitai Z

[West Coast Worm Meeting, 2000]

The netrin UNC-6 is involved in attraction and repulsion of circumferentially migrating cells and axons in C. elegans. Attraction to netrin/UNC-6 is mediated by the UNC-40 receptor, but the signalling events that occur upon UNC-40 activation remain unknown. In wild type animals UNC-6 is expressed ventrally, and several axons are directed ventrally via the UNC-40 receptor. This ventral migration is disrupted in unc-6 and unc-40 mutants. To probe the mechanism of UNC-40 activation by UNC-6, we deleted the extracellular domain of an unc-40 transgene and added a myristylation signal to the cytoplasmic domain, generating what we refer to as MYR::UNC-40. When expressed under the control of the mec-7 promoter, MYR::UNC-40 produced a variety of novel phenotypes in all mec-7-expressing touch cells. These phenotypes included exuberant axon outgrowth and branching, defective axon guidance, and enlarged and deformed cell bodies. The effects are independent of the endogenous UNC-6 ligand and UNC-40 receptor. These results lead us to believe that the MYR::UNC-40 represents a ligand-independent, constitutively active form of UNC-40. The generation of a gain-of-function UNC-40 molecule allowed us to search for downstream components of the UNC-40 signalling pathway by looking for mutations that suppressed the MYR::UNC-40-induced phenotype. One such mutation was in unc-115, a gene encoding a molecule with three LIM domains and an actin-binding villin headpiece. Both unc-40 and unc-115 mutants alone had defects in the ventral migration of the AVM axon. In the unc-40; unc-115 double mutant, the AVM axon guidance defect was not enhanced, again suggesting that UNC-115 might function in the UNC-40 pathway. The agreement of the loss-of-function double mutant phenotype with the MYR::UNC-40 suppression further supports the idea that MYR::UNC-40 represents a gain-of-function of UNC-40. Biochemical and cell biological experiments are currently underway to determine whether UNC-40 and UNC-115 physically associate. Structure/function experiments are also being conducted to determine the portion of the UNC-40 cytoplasmic domain responsible for signalling to UNC-115. Finally, we are continuing to search for new components of the UNC-40 signalling pathway by looking for additional suppressors of MYR::UNC-40.

Gitai Z et al. (2003) Neuron "The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM."

Gitai Z, Tessier-Lavigne M, Bargmann CI, Yu TW, Lundquist EA

[Neuron, 2003]

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.

Ketel, CS et al. (2005) Mol Cell Biol "Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes."

Vargas, ML, Andersen, EF, Strome, S, Ketel, CS, Suh, J, Simon, JA

[Mol Cell Biol, 2005]

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is > 1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Wan G et al. (2021) EMBO J "ZSP-1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans."

Pagano D, Dodson AE, Bajaj L, Kennedy S, Fei Y, Wan G, Fields B

[EMBO J, 2021]

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.

Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Krishna P et al. (1988) J Biol Chem "Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA."

Krishna P, Kennedy BP, van de Sande JH, McGhee JD

[J Biol Chem, 1988]

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Gorgon S et al. (2024) Cancer Res Commun "A drug discovery pipeline for MAPK/ERK pathway inhibitors in C. elegans."

Gorgon S, Hemmingsson O, Billing O, Eriksson AU

[Cancer Res Commun, 2024]

Oncogenic signaling through the MAPK/ERK pathway drives tumor progression in many cancers. While targeted MAPK/ERK pathway inhibitors improve survival in selected patients, most tumors are resistant. New drugs could be identified in small animal models that, unlike in vitro models, can address oral uptake, compound bioavailability and toxicity. This requires pharmacological conformity between human and model MAPK/ERK pathways, and available phenotypic assays. Here, we test if the conserved MAPK/ERK pathway in C. elegans could serve as a model for pharmacological inhibition and we develop in vivo pipelines for high throughput compound screens. Using fluorescence-based image analysis of vulva development as a readout for MAPK/ERK activity, we obtained excellent assay Z-scores for the MEK inhibitors trametinib (Z = 0.95), mirdametinib (Z = 0.93), and AZD8330 (Z = 0.87), as well as the ERK inhibitor temuterkib (Z = 0.86). The throughput was 800 wells per hour, with an average seed density of 25.5 animals per well. Readouts included drug efficacy, toxicity, and pathway specificity, which was tested against pathway activating upstream (lin-15)- and downstream (lin-1) mutants. To validate the model in a high throughput setting, we screened a blinded library of 433 anti-cancer compounds and identified four MEK inhibitors among seven positive hits. Our results highlight a high degree of pharmacological conformity between C. elegans and human MAPK/ERK pathways and the presented high-throughput pipeline may discover and characterize novel inhibitors in vivo.