Qin Z et al. (2018) G3 (Bethesda) "Genomic Identification and Functional Characterization of Essential Genes in Caenorhabditis elegans."

Chen N, Yu S, Johnsen R, Qin Z, Baillie D, Chu JS

[G3 (Bethesda), 2018]

Using combined genetic mapping, Illumina sequencing, bioinformatics analyses, and experimental validation, we identified 60 essential genes from 104 lethal mutations in two genomic regions of C. elegans totalling approximately 14 Mb on chromosome III(mid) and chromosome V(left). Five of the 60 genes had not previously been shown to have lethal phenotypes by RNA interference depletion. By analyzing the regions around the lethal missense mutations, we identified four putative new protein functional domains. Furthermore, functional characterization of the identified essential genes shows that most are enzymes, including helicases, tRNA synthetases, and kinases in addition to ribosomal proteins. Gene Ontology analysis indicated that essential genes often encode for enzymes that conduct nucleic acid binding activities during fundamental processes, such as intracellular DNA replication, transcription, and translation. Analysis of essential gene shows that they have fewer paralogs, encode proteins that are in protein interaction hubs, and are highly expressed relative to non-essential genes. All these essential gene traits in C. elegans are consistent with those of human disease genes. Most human orthologs (90%) of the essential genes in this study are related to human diseases. Therefore, functional characterization of essential genes underlines their importance as proxies for understanding the biological functions of human disease genes.

Qin Z et al. (2015) Nat Commun "Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells."

Hubbard EJ, Qin Z

[Nat Commun, 2015]

Stem cells maintain tissues and organs over the lifespan of individuals. How aging influences this process is unclear. Here we investigate the effects of aging on C. elegans germline stem/progenitor cells and show that the progenitor pool is depleted over time in a manner dependent on inhibition of DAF-16/FOXO by insulin/IGF-1 signalling (IIS). Our data indicate that DAF-16/FOXO activity in certain somatic gonad cells is required for germline progenitor maintenance, and that this role is separable from the effect of DAF-16/FOXO on organismal aging. In addition, blocking germ cell flux, similar to reducing IIS, maintains germline progenitors. This effect is partially dependent on gonadal DAF-16/FOXO activity. Our results imply that (1) longevity pathways can regulate aging stem cells through anatomically separable mechanisms, (2) stem cell maintenance is not necessarily prioritized and (3) stem cell regulation can occur at the level of an entire organ system such as the reproductive system.

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.

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.

Farkas Z et al. (2019) FASEB J "The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin."

Budai Z, Mehta A, Herit F, Shakir MA, Petric M, Zhou Z, Rajnavolgyi E, Takacs-Vellai K, Boissan M, Sandor S, Orban TI, Farkas Z, Kovacs T, Szondy Z, Jung SY, Niedergang F, Liu X, Qin J, Bajtay Z

[FASEB J, 2019]

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (<i>Caenorhabditis elegans</i>/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavolgyi, E., Szondy, Z., Budai, Z., Orban, T. I., Sandor, S., Mehta, A., Bajtay, Z., Kovacs, T., Jung, S. Y., Shakir, M. A., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takacs-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

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.

Henderson JR et al. (2003) Cell Motil Cytoskeleton "ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes."

Pomies P, Beckerle MC, Auffray C, Henderson JR

[Cell Motil Cytoskeleton, 2003]

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.

Mi-Mi L et al. (2012) J Cell Biol "Z-line formins promote contractile lattice growth and maintenance in striated muscles of C. elegans."

Mi-Mi L, Kemphues K, Pruyne D, Votra S, Bretscher A

[J Cell Biol, 2012]

Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line-associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.

Ono K et al. (2019) FEBS J "Kettin, the large actin-binding protein with multiple immunoglobulin domains, is essential for sarcomeric actin assembly and larval development in Caenorhabditis elegans."

Ono S, Qin Z, Johnsen RC, Baillie DL, Ono K

[FEBS J, 2019]

Among many essential genes in the nematode Caenorhabditis elegans, let-330 is located on the left arm of Chromosome V and was identified as the largest target of a mutagen in this region. However, the let-330 gene has not been characterized at the molecular level. Here, we report that two sequenced let-330 alleles are nonsense mutations of ketn-1, a previously characterized gene encoding kettin. Kettin is a large actin-binding protein of 472 kDa with 31 immunoglobulin domains and is expressed in muscle cells in C. elegans. let-330/ketn-1 mutants are homozygous lethal at the first larval stage with mild defects in body elongation. These mutants have severe defects in sarcomeric actin and myosin assembly in striated muscle. However, -actinin and vinculin, which are components of the dense bodies anchoring actin to the membranes, were not significantly disorganized by let-330/ketn-1 mutation. Kettin localizes to embryonic myofibrils before -actinin is expressed, and -actinin deficiency does not affect kettin localization in larval muscle. Depletion of vinculin minimally affects kettin localization but significantly reduces colocalization of actin with kettin in embryonic muscle cells. These results indicate that kettin is an essential protein for sarcomeric assembly of actin filaments in muscle cells. This article is protected by copyright. All rights reserved.