Kocsisova Z et al. (2018) J Vis Exp "Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU."

Mohammad A, Schedl T, Kocsisova Z, Kornfeld K

[J Vis Exp, 2018]

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Kocsisova Z et al. (2019) Development "Rapid, population-wide declines in stem cell number and activity during reproductive aging in C. elegans."

Kornfeld K, Schedl T, Kocsisova Z

[Development, 2019]

<i>C. elegans</i> hermaphrodites display dramatic age-related decline of reproduction early in life while somatic functions are still robust. To understand reproductive aging, we analyzed the assembly line of oocyte production that generates fertilized eggs. Aging germlines displayed both sporadic and population-wide changes. A small fraction of aging animals displayed endomitotic oocytes in the germline and other defects. By contrast, all animals displayed age-related decreases in germline size and function. As early as day 3 of adulthood, animals displayed fewer stem cells and a slower cell cycle, which combine to substantially decrease progenitor zone output. The <i>C. elegans</i> germline is the only adult tissue that contains stem cells, allowing the analysis of stem cells in aging. To investigate the mechanism of the decrease in stem cell number, we analyzed the Notch signaling pathway. The Notch effectors LST-1 and SYGL-1 displayed age-related decreases in expression domains, suggesting a role for Notch signaling in germline aging. The results indicate that while sporadic defects account for the sterility of some animals, population-wide changes account for the overall pattern of reproductive aging.

Kocsisova Z et al. (2018) BMC Dev Biol "Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans."

Kocsisova Z, Schedl T, Kornfeld K

[BMC Dev Biol, 2018]

BACKGROUND: The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase , has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. RESULTS: To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. CONCLUSIONS: In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

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.

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.

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.

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.