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[
Dev Cell,
2017]
In this issue of Developmental Cell, Dickinson etal. (2017) and Rodriguez etal. (2017), along with Wang etal. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C.elegans zygote.
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[
International Worm Meeting,
2017]
Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most well-studied entomopathogens, due to its broad lethality to diverse insect species, and to its effective commercial use as a biological control agent for insect pests. For this reason, it has become an important genetic model for studying parasitism, pathogenesis, and symbiosis. We used a newly published hybrid assembly pipeline to assemble the best genome of S. carpocapsae to date, comprising 86,259,276 bp in 86 scaffolds, with an N50 of 4.03 Mb, from a combination of 75X coverage Pacbio and 130X coverage Illumina reads. We found that 90% of the genome is represented by the 22 largest scaffolds. RNA-seq data from 17 developmental stages spanning the embryo to adult stages were used to help predict 22,295 gene models, a major reduction in the number of genes from the previously published assembly by Dillman et al. 2015, which has 28,313 genes, and an increase in the number of genes relative to a high contiguity S. carpocapsae Breton strain assembly (N50=1.24), which has 16,333 genes. Using this new genome, we infer the potential chromosomal origins of our scaffolds by comparing them to C. elegans using shared one-to-one orthologs and find that many of the largest scaffolds correspond primarily to single chromosomes in C. elegans. We also investigate a potential large 1.2 Mb duplication in the genome, and delve into gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations will provide a good foundation for new comparative genomic and gene expression studies.
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[
International Worm Meeting,
2017]
We are interested in using entomopathogenic nematodes (EPNs) from the family Steinernematidae, which are nematodes that parasitize and efficiently kill insects and are used as satellite model organism, to study the conservation of endoderm development in nematodes. The Steinernema carpocapsae genome lacks the GATA transcription factors END-1 and END-3, which control endoderm development in the E-cell of the 8-cell stage in C. elegans while their downstream target genes are conserved and expressed abundantly during endoderm development. Therefore, there must be an alternative set of early-expressed Transcription Factors(TFs) that determine endoderm cell fate in S. carpocapsae. We are isolating single-cells from S. carpocapsae and have sequenced individual single-cells from early stages of S. carpocapsae to identify early zygotic TFs that could be cell lineage specific. The embryonic localization of these TFs will be verified using single molecule fluorescent in situ hybridization(smFISH). We will compare our results in S. carpocapsae to the matching single-cell data from C. elegans E-cells to perform the first comparison of gene expression at the single-cell level among homologous cells across distant nematode species with a focus on regulatory genes controlling early endoderm development.
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Macchietto, Marissa, Murad, Rabi, Maya Rodriguez, Isaryhia, Serra, Lorrayne, Macias-Munoz, Aide, Mortazavi, Ali, Rodriguez, Bryan, Joan McGill, Cassandra
[
International Worm Meeting,
2019]
Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most studied entomopathogens due to its broad lethality to diverse insect species and its effective commercial use as a biological control agent for insect pests, as well as a genetic model for studying parasitism, pathogenesis, and symbiosis. In this study, we used long-reads from the Pacific Biosciences platform and BioNano Genomics Irys system to assemble the best genome of S. carpocapsae ALL strain to date, comprising 84.5 Mb in 16 scaffolds, with an N50 of 7.36Mb. The largest scaffold, with 20.9Mb, was identified as chromosome X based on sex-specific genome sequencing. The high level of contiguity allowed us to characterize gene density, repeat content, and GC content. RNA-seq data from 17 developmental stages, spanning from embryo to adult, were used to predict 30,957 gene models. Using this new genome, we performed a macrosyntenic analysis to Caenorhabditis elegans and Pristionchus pacificus and found S. carpocapsae's chromosome X to be primarily orthologous to C. elegans' and P. pacificus' chromosome II and IV. We also investigated the expansion of protein families and gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations provide a foundation for new comparative genomic and gene expression studies within the Steinernema clade and across the Nematoda phylum.
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[
J Biol Chem,
2001]
Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.
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[
FASEB J,
2007]
Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal (1). Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.--Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., Aviles, F. X. Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily.
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[
Methods Mol Biol,
2011]
In the past several years, a host of new technologies have made it possible to visualize single molecules within cells and organisms (Raj et al., Nat Methods 5:877-879, 2008; Pare et al., Curr Biol 19:2037-2042, 2009; Lu and Tsourkas, Nucleic Acids Res 37:
e100, 2009; Femino et al., Science 280:585-590, 1998; Rodriguez et al., Semin Cell Dev Biol 18:202-208, 2007; Betzig et al., Science 313:1642-1645, 2006; Rust et al., Nat Methods 3:793-796, 2006; Fusco et al., Curr Biol 13:161-167, 2003). Many of these are based on fluorescence, either fluorescent proteins or fluorescent dyes coupled to a molecule of interest. In many applications, the fluorescent signal is limited to a few pixels, which poses a classic signal processing problem: how can actual signal be distinguished from background noise? In this chapter, I present a MATLAB (MathWorks (2010) MATLAB. Retrieved from
http://www.mathworks.com) software suite designed to work with these single-molecule visualization technologies (Rifkin (2010) spotFinding Suite.
http://www.biology.ucsd.edu/labs/rifkin/software.html). It takes images or image stacks from a fluorescence microscope as input and outputs locations of the molecules. Although the software was developed for the specific application of identifying single mRNA transcripts in fixed specimens, it is more general than this and can be used and/or customized for other applications that produce localized signals embedded in a potentially noisy background. The analysis pipeline consists of the following steps: (a) create a gold-standard dataset, (b) train a machine-learning algorithm to classify image features as signal or noise depending upon user defined statistics, (c) run the machine-learning algorithm on a new dataset to identify mRNA locations, and (d) visually inspect and correct the results.
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[
International Worm Meeting,
2005]
Genes involved in coenzyme Q (Q) biosynthesis, such as
coq-7/clk-1, have regulatory functions in Caenorhabditis elegans aging and development. Silencing of C. elegans coq genes lead to life span extension (Asencio et al. 2003). Mutants of coq genes have been reported for
coq-7/clk-1 and
coq-3. Only
coq-7/clk-1 mutant strains have been established, whereas
coq-3 knockouts are lethal (Hihi et al. 2002). We show that
coq-8 knock-out animals developed until L3 larval stage and then arrested. Mutants did not develop mature gonads, and showed shortened life span under dietary Q-free conditions. We also constructed a COQ-8::GFP reporter under control of
coq-8 promoter. An age-dependent pattern of
coq-8 gene expression from the embryo to senescence is reported, providing genetic evidence for Q biosynthesis regulation during development and aging. COQ-8 expression started in a few blastomeres as early as of 8th embryo mitosis and increased during larval stages in several tissues, particularly in those with active bioenergetics such as muscle, nervous system and hypodermis throughout moulting period. Maximal COQ-8 expression was detected in L3 stage, matching with the maximal Q content (Jonassen et al. 2002), and the arresting stage in
coq-8 knockouts. Non-moulting young adults did not show hypodermis COQ-8 expression and in elderly individuals expression was detected only observed in nervous system. We propose a role for
coq-8 gene supporting the regulated Q biosynthesis that fulfils ubiquinone requirements in different tissues during life cycle in C. elegans. References: Asencio, C.; Rodriguez-Aguilera, J. C.; Ruiz-Ferrer, M.; Vela, J., and Navas, P. Silencing of ubiquinone biosynthesis genes extends life span in Caenorhabditis elegans. FASEB J. 2003 Jun; 17(9):1135-7. Hihi, A. K.; Gao, Y., and Hekimi, S. Ubiquinone is necessary for Caenorhabditis elegans development at mitochondrial and non-mitochondrial sites. J Biol Chem. 2002 Jan 18; 277(3):2202-6. Jonassen, T.; Marbois, B. N.; Faull, K. F.; Clarke, C. F., and Larsen, P. L. Development and fertility in Caenorhabditis elegans
clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria. J Biol Chem. 2002 Nov 22; 277(47):45020-7.
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[
International Worm Meeting,
2013]
Embryo development requires precise coordination of mechanical forces and their failure can lead to diseases. During the morphogenesis of C. elegans embryo, the cooperation of epidermal acto-myosin network and muscle contractions is essential. The acto-myosin activity in the epidermis, which has been shown to be more important in lateral than in dorsal-ventral cells, squeeze the embryo and make it elongate [1]. Muscle contractions become active around 1.7-1.8 fold stage. They have been showed to induce a mechano-transduction pathway [2], which is important for elongation. However, it is unclear how the contractions along the anterior-posterior axis help to increase the length of the embryo. Our project aims to elucidate the mechanical role of muscle contractions and its coordination with acto-myosin forces. The experiments are designed following a working model where muscle contractions induce a change in the elasticity of the embryo. We are using a laser nano-dissection technique to investigate cortical tension and elasticity of epidermal cells before and after the onset of muscle contractions. In parallel, we are evaluating the relative changes of acto-myosin forces with a FRET sensor [3] inserted in HMP-1 - a component of the adherens junctions. I will present our observations and preliminary results of the epidermal cortex nano-dissection experiments and measures of acto-myosin forces exerted on adherens junctions. References 1.Gally C, Wissler F, Zahreddine H, Quintin S, Landmann F, Labouesse M. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles. Development. 2009 Sep;136(18):3109-19. Epub 2009 Aug 12. 2.Zhang H, Landmann F, Zahreddine H, Rodriguez D, Koch M, Labouesse M. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature. 2011 Mar 3;471(7336):99-103. 3.Grashoff C, Hoffman B, Brenner M, Zhou R, Parsons M, Yang M, McLean M, Sligar S, Chen C, Ha T, Schwartz M. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010 July 8; 466(7303): 263-266.
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[
International Worm Meeting,
2011]
Cell polarity is crucial for the function of most cell types. Polarity genes have been identified in screens using single gene loss of function approaches. Due to partial redundancy and built in robustness of biological processes many polarity players and functional links between them are still missing. Some genes might have additional loss of function phenotypes masking their polarity role and making identification of genetic interactions between essential genes challenging. We have developed a large scale screening strategy to efficiently detect genetic interactions between essential genes using RNAi suppressor screens. Focusing screening on a sub-library of 2133 genes with embryonic functions, we carried out screens for 17 temperature sensitive (ts) embryonic lethal mutants. These were compromised for essential polarity functions such as actomyosin contractility, PAR protein localization and spindle positioning. We developed a high-throughput protocol to screen for suppressors of a mutant in less than 2 weeks. By modulating the penetrance of RNAi knockdown we have doubled the sensitivity of the suppressor screens for detecting essential genetic interactions. Overall, we tested over 50,000 interactions. Reproducible suppressors were considered specific if they failed to suppress five ts mutants not involved in polarity. The functional analysis of our interactions shows that genes involved in ribosomal functions and mitochondrial respiratory chain non-specifically suppress a broad variety of ts mutants and therefore have been excluded from our analysis.
We have built a cell polarity network with 186 genes connected by 229 different genetic interactions. The network shows a 3-fold enrichment for known cell polarity players and, for each mutant screened, confirmed interactions predicted from the literature. We observed that ts genes sharing polarity functions cluster with each other and we have identified new hubs linking actomyosin, PAR proteins and spindle positioning machinery. Importantly, specific profiles of interactions found in our network suggested functional roles for suppressors that we have been able to confirm experimentally. We have also used our network to predict functions for unknown proteins that share similar interactions with known cellular components (see Rodriguez et al., abstract).
Applying our screening strategy in other contexts should be a powerful way to identify key players and uncover novel mechanisms in other essential biological processes.